Transgenic t cell and chimeric antigen receptor t cell compositions and related methods

ABSTRACT

The invention provides a T cell wherein one or more therapeutic transgenes is integrated at a within the genome of the cell such that expression of the transgene is under control of an endogenous promoter of the T cell. The invention additional provides methods of making and using such cells to treat a subject with T cell therapy. The invention also provides a T cell wherein a recombinant nucleic acid sequence encoding a chimeric antigen receptor (CAR) is integrated at a first site within the genome of the cell such that the CAR is expressed by the cell at the surface of the cell, and wherein integration of the nucleic acid encoding the CAR at the first site reduces or prevents expression of a functional T cell receptor (TCR) complex at the surface of the cell. The invention additional provides methods of making and using such cells to treat a subject with CAR therapy.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional application No.62/323,623, filed Apr. 15, 2016, U.S. Provisional application No.62/323,675, filed Apr. 16, 2016, United States Provisional applicationNo. 62/461,677, filed Feb. 21, 2017, and U.S. Provisional applicationno. 62/462,243, filed Feb. 22, 2017, each of which is incorporated byreference herein in its entirety.

2. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Sequence Listing with thisapplication as an ASCII text file entitled “13542-043-228_SL.txt”created on Apr. 7, 2017, and having a size of 76,426 bytes.

3. FIELD

The present invention relates generally to immunotherapy, and morespecifically to immunotherapy using engineered immune cells such as Tcells.

4. BACKGROUND

Targeted immunotherapies rely on the use of immune cells or moleculesthat engage immune cells to treat a variety of diseases, includingcancer, infectious and autoimmune disorders (Miller & Sadelain, CancerCell. 27(4):439-49 (2015); Sabatos-Peyton et al., Curr. Opin. Immunol.22(5):609-615 (2010); McLeod & Anderton, Curr. Opin. Pharmacol. 23:1-108(2015)). Recently, the genetic modification of T cells to expresschimeric antigen receptors (CARs) that target tumor antigens has allowedthe successful eradication of leukemic cells in humans (Brentjens etal., Sci. Transl. Med. 5(177):177ra38. doi: 10.1126/scitranslmed.3005930(2013)). In the latter approach, the complementary DNA (cDNA) encodingthe CAR is delivered to T cells via integration-competentgamma-retroviruses or lentiviruses. These recombinant viral vectorsrequire a viral integrase enzyme, which catalyzes the integration of theviral DNA into the human genome in a semi-random manner (Schroder etal., Cell 110(4):521-529 (2002); Wu et al., Science 300(5626):1749-1751(2003)). A third approach makes use of a DNA transposition mechanism,whose components can be delivered into the cells without the need ofviral particles. In this case, a DNA transposase carries out theintegration of the DNA transposon (containing the gene(s) of interest)into the human genome, also in a semi-random fashion (Yant et al., Mol.Cell. Biol. 25(6):2085-2094 (2005)). All the above-mentioned genedelivery methods produce a T cell population exhibiting heterogeneousCAR expression due to different genomic locations of the integratedvector. This “variegated expression” limits the number of cells with aCAR expression that is optimal for target cell interaction and for Tcell activation strength. In addition, this uncontrolled DNA integrationmay potentially result in insertional mutagenesis, which can eitheractivate a proto-oncogene or inactivate a tumor suppressor gene. Anotherlimitation of these genetic modification approaches is that T cellsstill express their antigen receptor, known as TCR, which can stillparticipate in antigen recognition, thus activating the CART cell. Thispotential side effect limits the use of autologous CART cells inpatients with autoimmune disorders, or the use of allogeneic CAR T cellsin any recipient, two circumstances where the T cell may attack therecipient's tissues (causing autoimmunity in the first instance andgraft versus host disease (GvHD) in the latter).

The genetic modification of cells through homologous recombinationpermits the precise integration of exogenous DNA at chosen genomic sites(Cappechi et al., Nat. Rev. Genet. 6(6):507-512 (2005)). Such targeteddelivery has been recently described where a promoter-containing CARconstruct was targeted into the CCR5 locus in human primary T cells,which allowed the modified T cells to kill tumor cells in vitro (Satheret al., Sci. Transl. Med. 7(307):307ra156. doi:10.1126/scitranslmed.aac5530 (2015)). Though interesting, the authorsdid not show whether CAR expression driven by the MND promoter can bemaintained constant in the CCR5 locus, and more importantly, they didnot show that the level of CAR expression was optimal to eradicate tumorcells in vivo. In addition, CCR5 disruption has been linked to anincreased susceptibility to West Nile virus infection (Lim et al, TrendsImmunol. 27(7):308-312 (2006)).

Adoptive immunotherapy using chimeric antigen receptors (CARs) has shownremarkable clinical results in the treatment of leukemia and is one ofthe most promising new strategies to treat cancer. Current clinicalprotocols utilize autologous T cells that are collected by apheresis andengineered with retroviral vectors to stably express the CAR, which isresponsible for the recognition of an extracellular tumor molecule, andfor the activation of the engineered T cell. This approach requirespatient-specific cell manufacturing, which unavoidably results inpatient-to-patient variability in the final cell product. Widespreadimplementation of this approach will further require progress inautomation and miniaturization of cell manufacturing to meet the demandfor CAR T cells. Furthermore, current approaches utilize randomlyintegrating vectors, including gamma-retroviral, lentiviral andtransposons, which all result in semi-random integration and variableexpression of the CAR owing to transgene variegation. Position effectsmay result in heterogeneous T cell function, transgene silencing and,potentially, insertional oncogenesis. Thus, the conjunction ofautologous cell sourcing and random vector integration is prone togenerating cell products with variable potency.

Different tailored nucleases, including CRISPR/Cas9 system, Zinc FingerNucleases or TAL effector nucleases (TALENs), have been previously usedfor gene disruption in a wide range of human cells including primary Tcells. In some instances, these nucleases have been used to generateso-called “universal T cells” for allogeneic administration, bydisrupting T cell receptor (TCR) or HLA class I expression, but viralvectors or the sleeping beauty transposon were used to deliver the CARcDNA, all of which result in semi-random transgene integration and itsdownstream consequences.

To address the negative impact that TCR expression may have on thealloreactivity of CAR T cells, a number of laboratories have designedtailored nucleases (zinc-finger nuclease, TALE nuclease, and CRISPR/Cas9nuclease) that specifically target and cleave the 5′ end of the constantregion of the TCR alpha or beta chain (Provasi et al, Nat. Med.18(5):807-815 (2012); Poirot et al., Cancer Res. 75(18):3853-3864(2015); Osborn et al., Mol. Ther. 24(3):570-581 (2016)). The cleavage ateither site results in DNA modifications incorporated through the DNArepair mechanism called non-homologous end joining (NHEJ). The mutatedregion prevents correct splicing between the rearranged V(D)J genes withtheir respective constant region, thus impeding the proper assembly ofthe TCR complex at the cell surface. These TCR negative cells, whichwere disabled for causing GVHD, were then used to express CARs that weredelivered with transposons or lentiviruses (Torikai et al., Blood119(24):5697-5705 (2012); Poirot et al., Cancer Res. 75(18):3853-3864(2015)). Though these CART cells have the advantage of not expressing aTCR that can cause GVHD, they still present the above-mentioned pitfallsdue to the semi-random integration of the CAR gene (variegatedexpression, insertional mutagenesis).

Previously described approaches for genetically engineering a cell, suchas a T cell, include the use of inducible promoters within viral vectors(e.g., NFAT promoter in a retroviral vector, or synNotch constructs), orthe use of small molecules that control transcription or proteinaggregation (Ponomarev et al., Neoplasia 3(6):480-488 (2001); Zhang etal., Mol. Ther. 19(4): 751-759 (2011); Roybal et al., Cell167(2):419-432, e16 (2016): Wu et al., Science 16; 350(6258):aab4077(2015); Juillerat et al., Sci. report 18950 (2016)). These approachesare vulnerable to a number of complications and barriers, includingvariegated expression of randomly integrated transgenes, the necessityfor intravenous drug infusion and pharmacodynamic limitations of thesedrugs, and immunogenicity of some of the protein components used in someof these approaches (e.g., chimeric transcription factors, immunogenicprotein domains).

Chimeric antigen receptors (CARs) are synthetic receptors that redirectand reprogram T cells to mediate tumour rejection (Jensen et al., Curr.Opin. Immunol. 33:9-15 (2015)). The most successful CARs used to dateare those targeting CD19 (Brentjens et al., Nat. Med. 9:279-286 (2003)),which offer the prospect of complete remissions in patients withchemorefractory/relapsed B cell malignancies (Sadelain, J. Clin. Invest.125:3392-3400 (2015)). CARs are typically transduced into patient Tcells using γ-retroviral (Sadelain et al., Ninth InternationalImmunology Congress, Budapest, 88:34 (1992)) or other randomlyintegrating vectors (Wang et al., Mol. Ther. Oncolytics 3:16015 (2016)),which may result in clonal expansion, oncogenic transformation,variegated transgene expression and transcriptional silencing (Ellis,Hum. Gene. Ther. 16:1241-1246 (2005); Riviere et al., Blood119:1107-1116 (2012); von Kalle et al., Hum. Gene Ther. 25:475-481(2014)). Recent advances in genome editing enable efficientsequence-specific interventions in human cells (Wright et al., Cell164:29-44 (2016); Tsai et al., Nat. Rev. Genet. 17:300-312 (2016)),including targeted gene delivery to the CCR5 and AAVS1 loci (Lombardo etal., Nat. Methods 8:861-869 (2011); Sather et al., Sci. Transl. Med.7:307ra156 (2015)).

Thus, there exists a need for therapies to provide improved treatmentusing immunotherapy, such as treatment of cancer or other diseases. Thepresent invention satisfies this need.

5. SUMMARY OF THE INVENTION

The invention is reflected by the claims presented herein and asdescribed below. The present invention relates to T cells wherein atransgene is integrated within the genome of the T cells such thatexpression of the transgene is under the control of an endogenouspromoter, and to methods of using such cells.

In one aspect, provided herein is a T cell wherein a transgene isintegrated at a first site within the genome of the T cell such thatexpression of the transgene is under control of an endogenous promoterof the T cell, wherein the transgene encodes a therapeutic protein ortherapeutic nucleic acid. In certain embodiments, the transgene encodesa therapeutic protein. In certain embodiments, the transgene encodes atherapeutic nucleic acid. In certain embodiments, the transgene isintegrated at a single site within the genome. In certain embodiments,the transgene is integrated at two sites within the genome of the cell.In certain embodiments, the first site is an exon of the endogenous geneunder control of the endogenous promoter. In a particular embodiment,the first site is within the first exon of the endogenous gene.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, theendogenous promoter is constitutive. In certain embodiments, theendogenous promoter that is constitutive is selected from the groupconsisting of CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter,TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, and CD3zpromoter.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, theendogenous promoter is active in a subset of T cells. In certainembodiments, the endogenous promoter that is active in a subset of Tcells is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, CD3z promoter, actin promoter, CD25promoter, IL2 promoter, CD69 promoter, GzmB promoter, T-bet promoter,IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter,IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter,CD95 promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1promoter, CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DRpromoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter,CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter,CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter,Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter,IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, theendogenous promoter is inducible.

In certain embodiments, the endogenous promoter that is inducible isinduced by activation of the T cell. In certain embodiments, theendogenous promoter that is inducible is induced by binding of achimeric antigen receptor (CAR), a chimeric co-stimulatory receptor(CCR), T cell receptor (TCR), CD28, CD27, or 4-1BB expressed by the Tcell to its respective binding partner. In certain embodiments, thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner. In certain embodiments, thepromoter induced by binding of a CAR, CCR or TCR expressed by the T cellto its respective binding partner is selected from the group consistingof nuclear factor of activated T cells (NFAT) promoter, programmed death1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter,cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte-activationprotein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyteattenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand(FasL) promoter, TIGIT promoter, and 2B4 promoter.

In certain embodiments, the endogenous promoter that is inducible isinduced by binding of a ligand to an inhibitory receptor expressed bythe T cell. In certain embodiments where the promoter is induced bybinding of a ligand to an inhibitory receptor expressed by the T cell,the inhibitory receptor is selected from the group consisting of PD-1,CTLA4, TRAIL, LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4. In certainembodiments where the promoter is induced by binding of a ligand to aninhibitory receptor expressed by the T cell, the promoter is selectedfrom the group consisting of CPT1a promoter and ATGL promoter.

In certain embodiments, the endogenous promoter that is inducible isinduced by binding of a cytokine to a cytokine receptor expressed by theT cell. In certain embodiments where the promoter is induced by bindingof a cytokine to a cytokine receptor expressed by the T cell, thecytokine is selected from the group consisting of interleukin 2 (IL2),interleukin 7 (IL7), interleukin 15 (IL15), and interleukin 21 (IL21).In certain embodiments where the promoter is induced by binding of acytokine to a cytokine receptor expressed by the T cell, the cytokine isselected from the group consisting of interleukin 10 (IL10) andtransforming growth factor (TGFI3). In certain embodiments where thepromoter is induced by binding of a cytokine to a cytokine receptorexpressed by the T cell, the promoter is selected from the groupconsisting of T-bet promoter, Eomes promoter, GATA3 promoter, and CD45RApromoter.

In certain embodiments, the endogenous promoter that is inducible isinduced by contact of the cell with a nucleic acid. In certainembodiments where a promoter is induced by contact of the cell with anucleic acid, the nucleic acid is selected from the group consisting ofviral DNA, viral, RNA, and intracellular microRNA. In certainembodiments, where the promoter is induced by contact with a nucleicacid selected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA, the promoter is selected from the groupconsisting of Type I interferon (IFN) alpha, Type I IFN beta, IRF3,IRF7, NFkB, AP-1, TNF-alpha, IL1, and IL6.

In certain embodiments, the endogenous promoter that is inducible isinduced by contact of the cell with a metabolite. In certainembodiments, the metabolite is selected from the group consisting ofpyruvate, glutamine, and beta-hydroxybutyrate.

In certain embodiments, the endogenous promoter that is inducible isinduced by a metabolic change in the cell or contact of the cell with asubstance that causes a metabolic change in the cell. In a particularembodiment, the promoter induced by a metabolic change in the cell orcontact of the cell with a substance that causes a metabolic change inthe cell is PKM2 promoter.

In certain embodiments, the endogenous promoter that is inducible isinduced by a particular ion concentration in the cell or contact of thecell with a particular ion concentration. In certain embodiments, theion is potassium or calcium. In certain embodiments, the promoterinduced by a particular ion concentration in the cell or contact of thecell with a particular ion concentration is selected from the groupconsisting of IL2 promoter, TNFalpha promoter, and IFNgamma promoter.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, thetransgene encodes a molecule selected from the group consisting of aCAR, a CCR, a cytokine, a dominant negative, a microenvironmentmodulator, an antibody, a biosensor, a chimeric receptor ligand (CRL), achimeric immune receptor ligand (CIRL), a soluble receptor, a solutetransporter, an enzyme, a ribozyme, a genetic circuit, an epigeneticmodifier, a transcriptional activator, a transcriptional repressor, andnon-coding RNA.

In certain embodiments, the transgene encodes a cytokine. In oneembodiment, optionally the cytokine is immunostimulatory. In certainembodiments, the cytokine that is immunostimulatory is selected from thegroup consisting of IL2, IL12, IL15, and IL18. In another embodiment,optionally the cytokine is immunoinhibitory. In certain embodiments, thecytokine that is immunoinhibitory is selected from the group consistingof TGFBeta and IL10.

In certain embodiments, the transgene encodes an antibody. In certainembodiments, optionally the antibody is selected from the groupconsisting of an immunoglobulin, a Bi-specific T-cell engager (BiTE), adiabody, a dual affinity re-targeting (DART), a Fab, a F(ab′), a singlechain variable fragment (scFv), and a nanobody.

In certain embodiments, the transgene encodes a CAR. In a particularembodiment, the CAR binds to a cancer antigen.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, the Tcell is sensitized to a target antigen.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, atransgene (hereinafter “reporter transgene”) encoding a reportermolecule is integrated within the genome of the T cell such thatexpression of the reporter transgene is under control of a promoter,preferably an endogenous promoter of the T cell.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, the Tcell is derived from a human. In certain embodiments of a T cell derivedfrom a human, the T cell is a primary human T cell, a T cell derivedfrom a CD34 hematopoietic stem cell, a T cell derived from an embryonicstem cell, or a T cell derived from an induced pluripotent stem cell.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, thetransgene is integrated into the first site by targeted homologousrecombination. In certain embodiments, the targeted homologousrecombination is carried out by a method comprising using a zinc-fingernuclease (ZFN), a transcription activator-like effector nuclease(TALEN), a clustered regularly-interspersed short palindromic repeats(CRISPR) associated protein 9 (Cas9), Cpf1, pyrogen, Aureus,Meganuclease or a Mega-Tal.

In certain embodiments of a T cell wherein a transgene is integrated ata first site within the genome of the T cell as described above, thetransgene is integrated at a plurality of sites within the genome of theT cell, and such that expression of the transgene at the plurality ofsites is under the control of different endogenous promoters.

In another aspect, provided herein is a T cell wherein a first transgeneis integrated at a first site within the genome of the T cell such thatexpression of the first transgene is under control of a first endogenouspromoter of the T cell, and wherein a second transgene is integrated ata second site within the genome of the T cell, such that expression ofthe second transgene is under the control of a second endogenouspromoter, wherein the first and second endogenous promoters aredifferent promoters, and wherein the first transgene encodes a firsttherapeutic protein or first therapeutic nucleic acid, and the secondtransgene encodes a second therapeutic protein or second therapeuticnucleic acid, preferably wherein the first therapeutic protein or firsttherapeutic nucleic acid is different from the second therapeuticprotein or second therapeutic nucleic nucleic, respectively. In certainembodiments, the first transgene encodes a first therapeutic protein. Incertain embodiments, the first transgene encodes a first therapeuticnucleic acid. In certain embodiments, the second transgene encodes asecond therapeutic protein. In certain embodiments, the second transgeneencodes a second therapeutic nucleic acid.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, the firstendogenous promoter is constitutive, and the second endogenous promoteris inducible. In certain embodiments, the constitutive promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the first endogenous promoter and/or the secondendogenous promoter is active in a subset of T cells. In certainembodiments, the first endogenous promoter and/or the second endogenouspromoter is independently selected from the group consisting of CD4promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter,CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, actinpromoter, CD25 promoter, IL2 promoter, CD69 promoter, GzmB promoter,T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3promoter, IL5 promoter, IL13 promoter, IL10 promoter, IL17A promoter,IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter,CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD127promoter, PD-1 promoter, CD122 promoter, CD132 promoter, KLRG-1promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter,CD11a promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD103promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter,CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme Apromoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD161promoter, IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by activationof the T cell.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by binding of achimeric antigen receptor (CAR), a chimeric co-stimulatory receptor(CCR), T cell receptor (TCR), CD28, CD27, and 4-1BB expressed by the Tcell to its respective binding partner. In certain embodiments, theinducible promoter is induced by binding of a CAR, CCR or TCR expressedby the T cell to its respective binding partner. In certain embodimentswhere the inducible promoter is induced by binding of a CAR, CCR or TCRexpressed by the T cell to its respective binding partner, the induciblepromoter is selected from the group consisting of nuclear factor ofactivated T cells (NFAT) promoter, programmed death 1 (PD-1) promoter, Tcell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyteantigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3)promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and2B4 promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by binding of aligand to an inhibitory receptor expressed by the T cell. In certainembodiments, the inhibitory receptor is selected from the groupconsisting of PD-1, CTLA4, TRAIL, LAG-3, BTLA, TIM-3, Fas, TIGIT, and2B4. In certain embodiments, the inducible promoter is selected from thegroup consisting of CPT1a promoter and ATGL promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by binding of acytokine to a cytokine receptor expressed by the T cell. In certainembodiments, the cytokine is selected from the group consisting ofinterleukin 2 (IL2), interleukin 7 (IL7), interleukin 15 (IL15), andinterleukin 21 (IL21). In certain embodiments, the cytokine is selectedfrom the group consisting of interleukin 10 (IL10) and transforminggrowth factor β (TGFβ). In certain embodiments, the inducible promoteris selected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by contact ofthe cell with a nucleic acid. In certain embodiments, the nucleic acidis selected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA. In certain embodiments where the induciblepromoter is induced by contact of the cell with viral DNA, viral, RNA,or intracellular microRNA, the inducible promoter is selected from thegroup consisting of Type I interferon (IFN) alpha, Type I IFN beta,IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1 and IL6.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by contact ofthe cell with a metabolite. In certain embodiments, the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by a metabolicchange in the cell or contact of the cell with a substance that causes ametabolic change in the cell. In certain embodiments, such an induciblepromoter is PKM2 promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the inducible promoter is induced by a particularion concentration in the cell or contact of the cell with a particularion concentration. In certain embodiments, the ion is potassium orcalcium. In certain embodiments where the inducible promoter is inducedby a particular ion concentration in the cell or contact of the cellwith a particular ion concentration, the inducible promoter is selectedfrom the group consisting of IL2 promoter, TNFalpha promoter, andIFNgamma promoter.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the first transgene and/or second transgene each encodes amolecule independently selected from the group consisting of a CAR, aCCR, a cytokine, a dominant negative, a microenvironment modulator, anantibody, a biosensor, a chimeric receptor ligand (CRL), a chimericimmune receptor ligand (CIRL), a soluble receptor, a solute transporter,an enzyme, a ribozyme, a genetic circuit, an epigenetic modifier, atranscriptional activator, a transcriptional repressor, and non-codingRNA.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the first transgene and/or second transgene encodes a cytokine.In certain embodiments wherein the first and/or second transgene encodesa cytokine, the cytokine preferably is immunostimulatory. In certainembodiments where the cytokine is immunostimulatory, the cytokine isselected from the group consisting of IL2, IL12, IL15, and IL18. Incertain embodiments wherein the first transgene and/or second transgeneencodes a cytokine, the cytokine preferably is immunoinhibitory. Incertain embodiments where the cytokine is immunoinhibitory, the cytokineis selected from the group consisting of TGFBeta and IL10.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the first transgene and/or second transgene encodes an antibody.In certain embodiments, the antibody is an immunoglobulin, a Bi-specificT-cell engager (BiTE), a diabody, a dual affinity re-targeting (DART), aFab, a F(ab′), a single chain variable fragment (scFv), and a nanobody.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the first transgene and/or second transgene encodes a CAR. In aparticular embodiment, the CAR binds to a cancer antigen.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the T cell is sensitized to a target antigen.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, a transgene (hereinafter “reporter transgene”) encoding areporter molecule is integrated within the genome of the T cell suchthat expression of the reporter transgene is under control of apromoter, preferably an endogenous promoter of the T cell.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the T cell is derived from a human. In certain embodiments of a Tcell derived from a human, the T cell is a primary human T cell, a Tcell derived from a CD34 hematopoietic stem cell, a T cell derived froman embryonic stem cell, or a T cell derived from an induced pluripotentstem cell.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the first transgene and/or second transgene is integrated intothe first site by targeted homologous recombination. In certainembodiments, the targeted homologous recombination is carried out by amethod comprising using a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), a clusteredregularly-interspersed short palindromic repeats (CRISPR) associatedprotein 9 (Cas9), Cpf1, pyrogen, Aureus, Meganuclease or a Mega-Tal.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, as describedabove, the first therapeutic protein or first therapeutic nucleic acidis different from said second therapeutic protein or second therapeuticnucleic nucleic, respectively.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the second endogenous promoter is induced byactivation of the T cell.

In certain embodiments of a T cell wherein a first transgene isintegrated at a first site within the genome and a second transgene isintegrated at a second site within the genome of the cell, and whereinthe first endogenous promoter is constitutive, and the second endogenouspromoter is inducible, the first transgene encodes a CAR. In aparticular embodiment where the first transgene encodes a CAR, the firstendogenous promoter is a T cell receptor promoter. In certainembodiments, the T cell receptor promoter is selected from the groupconsisting of T cell receptor alpha chain promoter, T cell receptor betachain promoter, CD3 gamma chain promoter, CD3 delta chain promoter, CD3epsilon chain promoter, and CD3 zeta chain promoter. In a particularembodiment, the promoter is T cell receptor alpha chain promoter.

In certain embodiments of a T cell described above, except insofar asthe foregoing embodiments relate to a transgene encoding a cytokine thatis immunoinhibitory, for example, TGFbeta or IL10, the T cell is animmunostimulatory T cell. In certain embodiments where the T cell is animmunoinhibitory T cell, the T cell is selected from the groupconsisting of cytotoxic T lymphocyte (CTL), CD4+ subtype, CD8+ subtype,central memory T cell (TCM), stem memory T cell (TSCM), effector memoryT cell, effector T cell, Th1 cell, Th2 cell, Th9 cell, Th17 cell, Th22cell, and Tfh (follicular helper) cell. In a specific embodiment, the Tcell is CD4+. In a specific embodiment, the T cell is CD8+.

In certain embodiments of a T cell described above, except insofar asthe foregoing embodiments relate to a transgene encoding a cytokine thatis immunostimulatory, for example, the cytokine is selected from thegroup consisting of IL2, IL12, IL15, and IL18, the T cell is animmunoinhibitory T cell. In a specific embodiment, the T cell is aregulatory T cell.

In another aspect, provided herein is an isolated population of T cells,which comprises a plurality of the T cell of the embodiments describedabove. In certain embodiments, the isolated population of T cellscomprises the immunostimulatory T cells described above. In certainembodiments, the isolated population of T cells comprises theimmunoinhibitory T cells described above.

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the T cell of theembodiments described above; and a pharmaceutically acceptable carrier.In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of a population of Tcells, which population comprises a plurality of the T cell of theembodiments described above; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the T cell of theembodiments described above, wherein the T cell is the immunostimulatoryT cell described above; and a pharmaceutically acceptable carrier. Inanother aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of a population of Tcells, which population comprises a plurality of the T cell of theembodiments described above, wherein the T cell is the immunostimulatoryT cell described above; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the T cell of theembodiments described above, wherein the T cell is the immunoinhibitorycell described above; and a pharmaceutically acceptable carrier. Inanother aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of a population of Tcells, which population comprises a plurality of the T cell of theembodiments described above, wherein the T cell is the immunoinhibitoryT cell described above; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating a subjectwith T cell therapy in need thereof, comprising administering to thesubject a therapeutically effective amount of the T cell of theembodiments described above. In another aspect, also provided herein isa method of treating a subject with T cell therapy in need thereof,comprising administering to the subject a therapeutically effectiveamount of the T cell population of the embodiments described above. Inyet another aspect, provided herein is a method of treating a subjectwith T cell therapy in need thereof, comprising administering to thesubject the pharmaceutical composition of the embodiments describedabove.

In certain embodiments of methods of the invention described above, thesubject is a human, and the T cell is derived from a human. In certainembodiments of the methods of the invention described above, the T cellis autologous to the subject. In certain embodiments of the methods ofthe invention described above, the T cell is non-autologous to thesubject.

In another aspect, provided herein is a method of treating a subjectwith T cell therapy in need thereof, wherein the subject is in need of astimulated immune response, comprising administering to the subject atherapeutically effective amount of a cell or population of cells,wherein the cell is a T cell, wherein a transgene is integrated at afirst site within the genome of the T cell such that expression of thetransgene is under control of an endogenous promoter of the T cell,wherein the transgene encodes a therapeutic protein or therapeuticnucleic acid. In certain embodiments, the cell or cell population isadministered to the subject as a pharmaceutical composition. In certainembodiments, the transgene encodes a therapeutic protein. In certainembodiments, the transgene encodes a therapeutic nucleic acid.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response as described above, the transgene is integrated at asingle site within the genome. In certain embodiments, wherein thetransgene is integrated at two sites within the genome of the cell. Incertain embodiments, wherein the first site is an an exon of theendogenous gene under control of the endogenous promoter. In a specificembodiment, the first site is within the first exon of the endogenousgene.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response as described above, the endogenous promoter isconstitutive. In certain embodiments, the constitutive promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response as described above, the endogenous promoter is active ina subset of T cells. In certain embodiments where the endogenouspromoter is active in a subset of T cells, the endogenous promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, CD3z promoter, actin promoter, CD25 promoter, IL2promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgammapromoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5 promoter,IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter, IL21promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter,PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1 promoter,CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DR promoter, CD38promoter, CD69 promoter, Ki-67 promoter, CD11a promoter, CD58 promoter,CD99 promoter, CD62L promoter, CD103 promoter, CCR4 promoter, CCR5promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter,CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter,Perforin promoter, CD57 promoter, CD161 promoter, IL-18Ra promoter,c-Kit promoter, and CD130 promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response as described above, the endogenous promoter isinducible.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the endogenous promoter is induced by activation of the T cell.In certain embodiments where the endogenous promoter is induced byactivation of the T cell, the promoter is induced by binding of achimeric antigen receptor (CAR), a chimeric co-stimulatory receptor(CCR), T cell receptor (TCR), CD28, CD27, or 4-1BB expressed by the Tcell to its respective binding partner. In certain embodiments, thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner. In certain embodiments where thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner, the promoter is selected fromthe group consisting of nuclear factor of activated T cells (NFAT)promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulinmucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4)promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter,CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and 2B4promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by binding of a ligand to an inhibitoryreceptor expressed by the T cell. In certain embodiments, the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4. In certain embodiments wherethe promoter is induced by binding of a ligand to an inhibitory receptorexpressed by the T cell, the promoter is selected from the groupconsisting of CPT1a promoter and ATGL promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by binding of a cytokine to a cytokinereceptor expressed by the T cell. In certain embodiments, the cytokineis selected from the group consisting of interleukin 2 (IL2),interleukin 7 (IL7), interleukin 15 (IL 15), and interleukin 21 (IL21).In certain embodiments where the promoter is induced by binding of acytokine to a cytokine receptor expressed by the T cell, the promoter isselected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by contact of the cell with a nucleicacid. In certain embodiments, the nucleic acid is selected from thegroup consisting of viral DNA, viral, RNA, and intracellular microRNA.In certain embodiments where the promoter is induced by contact of thecell with a nucleic acid selected from the group consisting of viralDNA, viral, RNA, and intracellular microRNA, the promoter is selectedfrom the group consisting of Type I interferon (IFN) alpha, Type I IFNbeta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, ILL and IL6.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by contact of the cell with a metabolite.In certain embodiments, the metabolite is selected from the groupconsisting of pyruvate, glutamine, and beta-hydroxybutyrate.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by a metabolic change in the cell orcontact of the cell with a substance that causes a metabolic change inthe cell. In a specific embodiment where the promoter is induced by ametabolic change in the cell or contact of the cell with a substancethat causes a metabolic change in the cell, the promoter is PKM2promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by a particular ion concentration in thecell or contact of the cell with a particular ion concentration. Incertain embodiments, the ion is potassium or calcium. In certainembodiments the promoter is induced by a particular ion concentration inthe cell or contact of the cell with a particular ion concentration, thepromoter is selected from the group consisting of IL2 promoter, TNFalphapromoter, and IFNgamma promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the transgene encodes a moleculeselected from the group consisting of a CAR, a CCR, a cytokine, adominant negative, a microenvironment modulator, an antibody, abiosensor, a chimeric receptor ligand (CRL), a chimeric immune receptorligand (CIRL), a soluble receptor, a solute transporter, an enzyme, aribozyme, a genetic circuit, an epigenetic modifier, a transcriptionalactivator, a transcriptional repressor, and non-coding RNA.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the transgene encodes a cytokine.In certain embodiments, optionally the cytokine is immunostimulatory. Incertain embodiments where the cytokine is immunostimulatory, thecytokine is selected from the group consisting of IL2, IL12, IL15, andIL18.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the transgene encodes an antibody.In certain embodiments, optionally the antibody is selected from thegroup consisting of an immunoglobulin, a Bi-specific T-cell engager(BiTE), a diabody, a dual affinity re-targeting (DART), a Fab, a F(ab′),a single chain variable fragment (scFv), and a nanobody.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the transgene encodes a CAR. In aspecific embodiment, the CAR binds to a cancer antigen.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the T cell is sensitized to atarget antigen.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, a transgene (hereinafter “reportertransgene”) encoding a reporter molecule is integrated within the genomeof the T cell such that expression of the reporter transgene is undercontrol of a promoter. Preferably, such a promoter is an endogenouspromoter of the T cell.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the T cell is derived from a human.In certain embodiments, the T cell is a primary human T cell, a T cellderived from a CD34 hematopoietic stem cell, a T cell derived from anembryonic stem cell, or a T cell derived from an induced pluripotentstem cell.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the transgene is integrated intothe first site by targeted homologous recombination. IN certainembodiments, the targeted homologous recombination is carried out by amethod comprising using a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), a clusteredregularly-interspersed short palindromic repeats (CRISPR) associatedprotein 9 (Cas9), Cpf1, pyrogen, Aureus, Meganuclease or a Mega-Tal.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the transgene is integrated at aplurality of sites within the genome of the T cell, and such thatexpression of the transgene at the plurality of sites is under thecontrol of different endogenous promoters.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the T cell is an immunostimulatoryT cell. In certain embodiments, the T cell is selected from the groupconsisting of cytotoxic T lymphocyte (CTL), CD4+ subtype, CD8+ subtype,central memory T cell (TCM), stem memory T cell (TSCM), effector memoryT cell, effector T cell, Th1 cell, Th2 cell, Th9 cell, Th17 cell, Th22cell, and Tfh (follicular helper) cell. In a specific embodiment, the Tcell is CD4+. In another specific embodiment, the T cell is CD8+.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above the subject has cancer. In aspecific embodiment, the cancer is leukemia.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the subject has a tumor.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the subject is a human, and the Tcell is derived from a human.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of a stimulatedimmune response, as described above, the T cell is autologous to thesubject. In certain embodiments of the methods of treating a subjectwith T cell therapy in need thereof, wherein the subject is in need of astimulated immune response, as described above, the T cell isnon-autologous to the subject.

In another aspect, provided herein is a method of treating a subjectwith T cell therapy in need thereof, wherein the subject is in need ofan inhibited immune response, comprising administering to the subject atherapeutically effective amount of a cell or population of cells,wherein the cell is a T cell, wherein a transgene is integrated at afirst site within the genome of the T cell such that expression of thetransgene is under control of an endogenous promoter of the T cell,wherein the transgene encodes a therapeutic protein or therapeuticnucleic acid. In certain embodiments, the cell or cell population isadministered as a pharmaceutical composition. In certain embodiments,the transgene encodes a therapeutic protein. In certain embodiments, thetransgene encodes a therapeutic nucleic acid.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene is integrated at asingle site within the genome. In certain embodiments, the transgene isintegrated at two sites within the genome of the cell. In certainembodiments, the first site is an an exon of the endogenous gene undercontrol of the endogenous promoter. In a particular embodiment, thefirst site is within the first exon of the endogenous gene.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the endogenous promoter isconstitutive. In certain embodiments, the constitutive promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the endogenous promoter is activein a subset of T cells. In certain embodiments, the endogenous promoterthat is active in a subset of T cells is selected from the groupconsisting of CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter,TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3zpromoter, actin promoter, CD25 promoter, IL2 promoter, CD69 promoter,GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4promoter, GATA3 promoter, IL5 promoter, IL13 promoter, IL10 promoter,IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter,CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27promoter, CD127 promoter, PD-1 promoter, CD122 promoter, CD132 promoter,KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67promoter, CD11a promoter, CD58 promoter, CD99 promoter, CD62L promoter,CD103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter,Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57promoter, CD161 promoter, IL-18Ra promoter, c-Kit promoter, and CD130promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the endogenous promoter isinducible.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the endogenous promoter is induced by activation of the T cell.In certain embodiments where the endogenous promoter is induced byactivation of the T cell, the promoter is induced by binding of achimeric antigen receptor (CAR), a chimeric co-stimulatory receptor(CCR), T cell receptor (TCR), CD28, CD27, or 4-1BB expressed by the Tcell to its respective binding partner. In certain embodiments, thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner. In certain embodiments where thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner, the promoter is selected fromthe group consisting of nuclear factor of activated T cells (NFAT)promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulinmucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4)promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter,CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and 2B4promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by binding of a ligand to an inhibitoryreceptor expressed by the T cell. In certain embodiments, the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4. In certain embodiments wherethe promoter is induced by binding of a ligand to an inhibitory receptorexpressed by the T cell, the promoter is selected from the groupconsisting of CPT1a promoter and ATGL promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by binding of a cytokine to a cytokinereceptor expressed by the T cell. In certain embodiments, the cytokineis selected from the group consisting of interleukin 10 (IL10) andtransforming growth factor β (TGFβ). In certain embodiments where thepromoter is induced by binding of a cytokine to a cytokine receptorexpressed by the T cell, the promoter is selected from the groupconsisting of T-bet promoter, Eomes promoter, GATA3 promoter, and CD45RApromoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by contact of the cell with a nucleicacid. In certain embodiments, the nucleic acid is selected from thegroup consisting of viral DNA, viral, RNA, and intracellular microRNA.In certain embodiments where the promoter is induced by contact of thecell with a nucleic acid selected from the group consisting of viralDNA, viral, RNA, and intracellular microRNA, the promoter is selectedfrom the group consisting of Type I interferon (IFN) alpha, Type I IFNbeta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1, and IL6.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by contact of the cell with a metabolite.In certain embodiments, the metabolite is selected from the groupconsisting of pyruvate, glutamine, and beta-hydroxybutyrate.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by a metabolic change in the cell orcontact of the cell with a substance that causes a metabolic change inthe cell. In certain embodiments where the promoter is induced by ametabolic change in the cell or contact of the cell with a substancethat causes a metabolic change in the cell, the promoter is PKM2promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response and the endogenous promoter is inducible, as describedabove, the promoter is induced by a particular ion concentration in thecell or contact of the cell with a particular ion concentration. Incertain embodiments, the ion is potassium or calcium. In certainembodiments where the promoter is induced by a particular ionconcentration in the cell or contact of the cell with a particular ionconcentration, the promoter is selected from the group consisting of IL2promoter, TNFalpha promoter, and IFNgamma promoter.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene encodes a moleculeselected from the group consisting of a CAR, a CCR, a cytokine, adominant negative, a microenvironment modulator, an antibody, abiosensor, a chimeric receptor ligand (CRL), a chimeric immune receptorligand (CIRL), a soluble receptor, a solute transporter, an enzyme, aribozyme, a genetic circuit, an epigenetic modifier, a transcriptionalactivator, a transcriptional repressor, and non-coding RNA.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene encodes a cytokine.In certain embodiments, optionally the cytokine is immunoinhibitory. Incertain embodiments, the cytokine that is immunoinhibitory is selectedfrom the group consisting of TGFBeta and IL10.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene encodes an antibody.In certain embodiments, optionally the antibody is selected from thegroup consisting of an immunoglobulin, a Bi-specific T-cell engager(BiTE), a diabody, a dual affinity re-targeting (DART), a Fab, a F(ab′),a single chain variable fragment (scFv), and a nanobody.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene encodes a CAR. In aspecific embodiment, the CAR binds to a cancer antigen.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the T cell is sensitized to atarget antigen.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, a transgene (hereinafter “reportertransgene”) encoding a reporter molecule is integrated within the genomeof the T cell such that expression of the reporter transgene is undercontrol of a promoter. Preferably, the reporter is under control of anendogenous promoter of the T cell.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the T cell is derived from a human.In certain embodiments, the T cell is a primary human T cell, a T cellderived from a CD34 hematopoietic stem cell, a T cell derived from anembryonic stem cell, or a T cell derived from an induced pluripotentstem cell.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene is integrated intothe first site by targeted homologous recombination. In certainembodiments, the targeted homologous recombination is carried out by amethod comprising using a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), a clusteredregularly-interspersed short palindromic repeats (CRISPR) associatedprotein 9 (Cas9), Cpf1, pyrogen, Aureus, Meganuclease or a Mega-Tal.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the transgene is integrated at aplurality of sites within the genome of the T cell, and such thatexpression of the transgene at said plurality of sites is under thecontrol of different endogenous promoters.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the T cell is an immunoinhibitory Tcell. In a specific embodiment, the immunoinhibitory T cell is aregulatory T cell.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the subject is a human, and the Tcell is derived from a human.

In certain embodiments of the methods of treating a subject with T celltherapy in need thereof, wherein the subject is in need of an inhibitedimmune response, as described above, the cell is autologous to thesubject. In certain embodiments of the methods of treating a subjectwith T cell therapy in need thereof, wherein the subject is in need ofan inhibited immune response, as described above, the cell isnon-autologous to the subject.

In another aspect, provided herein is a method of generating a T cellthat expresses a therapeutic transgene, comprising: introducing into a Tcell: (i) a transgene, and (ii) a homologous recombination systemsuitable for targeted integration of the transgene at a site within thegenome of the cell, whereby the homologous recombination systemintegrates the transgene at the site within the genome of the cell, andwherein expression of the transgene is under the control of anendogenous promoter, wherein the transgene encodes a therapeutic proteinor a therapeutic nucleic acid. In certain embodiments, the transgeneencodes a therapeutic protein. In certain embodiments, the transgeneencodes a therapeutic nucleic acid.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the endogenouspromoter is constitutive. In certain embodiments, the endogenousconstitutive promoter is selected from the group consisting of CD4promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter,CD3d promoter, CD3g promoter, CD3e promoter, and CD3z promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the endogenouspromoter is active in a subset of T cells. In certain embodiments wherethe endogenous promoter is active in a subset of T cells, the endogenouspromoter is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, CD3z promoter, actin promoter, CD25promoter, IL2 promoter, CD69 promoter, GzmB promoter, T-bet promoter,IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter,IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter,CD95 promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1promoter, CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DRpromoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter,CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter,CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter,Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter,IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the endogenouspromoter is inducible.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the endogenous promoter is induced byactivation of the T cell.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by binding of achimeric antigen receptor (CAR), a chimeric co-stimulatory receptor(CCR), T cell receptor (TCR), CD28, CD27, and 4-1BB expressed by the Tcell to its respective binding partner. In certain embodiments, thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner. In certain embodiments where thepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner, the promoter is selected fromthe group consisting of nuclear factor of activated T cells (NFAT)promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulinmucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4)promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter,CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and 2B4promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by binding of aligand to an inhibitory receptor expressed by the T cell. In certainembodiments, the inhibitory receptor is selected from the groupconsisting of PD-1, CTLA4, TRAIL, LAG-3, BTLA, TIM-3, Fas, TIGIT, and2B4. In certain embodiments where the promoter is induced by binding ofa ligand to an inhibitory receptor expressed by the T cell, the promoteris selected from the group consisting of CPT1a promoter and ATGLpromoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by binding of acytokine to a cytokine receptor expressed by the T cell. In certainembodiments where the promoter is induced by binding of a cytokine to acytokine receptor expressed by the T cell, the cytokine is selected fromthe group consisting of interleukin 2 (IL2), interleukin 7 (IL7),interleukin 15 (IL15), and interleukin 21 (IL21). In certain embodimentswhere the promoter is induced by binding of a cytokine to a cytokinereceptor expressed by the T cell, the cytokine is selected from thegroup consisting of interleukin 10 (IL10) and transforming growth factorβ (TGFβ). In certain embodiments where the promoter is induced bybinding of a cytokine to a cytokine receptor expressed by the T cell,the promoter is selected from the group consisting of T-bet promoter,Eomes promoter, GATA3 promoter, and CD45RA promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by contact of thecell with a nucleic acid. In certain embodiments, the nucleic acid isselected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA. In certain embodiments where the promoter isinduced by contact of the cell with a nucleic acid selected from thegroup consisting of viral DNA, viral, RNA, and intracellular microRNA,the promoter is selected from the group consisting of Type I interferon(IFN) alpha, Type I IFN beta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1,and IL6.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by contact of thecell with a metabolite. In certain embodiments, the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by a metabolicchange in the cell or contact of the cell with a substance that causes ametabolic change in the cell. In certain embodiments where the promoteris induced by a metabolic change in the cell or contact of the cell witha substance that causes a metabolic change in the cell, the promoter isPKM2 promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene and where the endogenous promoter isinducible, as described above, the promoter is induced by a particularion concentration in the cell or contact of the cell with a particularion concentration. In certain embodiments, the ion is potassium orcalcium. In certain embodiments where the promoter is induced by aparticular ion concentration in the cell or contact of the cell with aparticular ion concentration, the promoter is selected from the groupconsisting of IL2 promoter, TNFalpha promoter, and IFNgamma promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgeneencodes a molecule selected from the group consisting of a CAR, a CCR, acytokine, a dominant negative, a microenvironment modulator, anantibody, a biosensor, a chimeric receptor ligand (CRL), a chimericimmune receptor ligand (CIRL), a soluble receptor, a solute transporter,an enzyme, a ribozyme, a genetic circuit, an epigenetic modifier, atranscriptional activator, a transcriptional repressor, and non-codingRNA.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgeneencodes a cytokine. In certain embodiments, optionally the cytokine isimmunostimulatory. In certain embodiments, the immunostimulatorycytokine is selected from the group consisting of IL2, IL12, IL15, andIL18. In certain embodiments, optionally the cytokine isimmunoinhibitory. In certain embodiments, the immunoinhibitory cytokineis selected from the group consisting of TGFBeta and IL10.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgeneencodes an antibody. In certain embodiments, optionally the antibody isselected from the group consisting of an immunoglobulin, a Bi-specificT-cell engager (BiTE), a diabody, a dual affinity re-targeting (DART), aFab, a F(ab′), a single chain variable fragment (scFv), and a nanobody.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgeneencodes a CAR. In a specific embodiment, the CAR binds to a cancerantigen.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the T cell issensitized to a target antigen.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, a transgene(hereinafter “reporter transgene”) encoding a reporter molecule isintegrated within the genome of the T cell such that expression of thereporter transgene is under control of a promoter, preferably anendogenous promoter of the T cell.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the T cell isderived from a human. In certain embodiments, the T cell is a primaryhuman T cell, a T cell derived from a CD34 hematopoietic stem cell, a Tcell derived from an embryonic stem cell, or a T cell derived from aninduced pluripotent stem cell.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgene isintegrated into the first site by targeted homologous recombination. Incertain embodiments, the targeted homologous recombination is carriedout by a method comprising using a zinc-finger nuclease (ZFN), atranscription activator-like effector nuclease (TALEN), a clusteredregularly-interspersed short palindromic repeats (CRISPR) associatedprotein 9 (Cas9), Cpf1, pyrogen, Aureus, Meganuclease or a Mega-Tal.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgene isintegrated at a plurality of sites within the genome of the T cell, andsuch that expression of the transgene at said plurality of sites isunder the control of different endogenous promoters.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgenethat is introduced into the cell is contained in a targeting construct.In certain embodiments, the targeting construct comprises viral nucleicacid sequences. In certain embodiments, the targeting construct ispackaged into a natural or recombinant adeno-associated virus (AVV)viral particle. In a specific embodiment, the AAV particle comprisesAAV6 sequences. In certain embodiments, the targeting construct ispackaged into a non-integrating gamma-retrovirus.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the transgene inthe targeting construct is not operably linked to a promoter.

In certain embodiments of the methods of generating a T cell thatexpresses a therapeutic transgene, as described above, the methodfurther comprising introducing a second transgene into the T cell. Incertain embodiments, the first transgene is under control of anendogenous constitutive promoter and the second transgene is undercontrol of an endogenous inducible promoter. In certain embodiments, thefirst transgene is a CAR. In certain embodiments where the transgene isa CAR, the endogenous constitutive promoter is a T cell receptorpromoter. In certain embodiments where the promoter is a T cell receptorpromoter, the promoter is selected from the group consisting of T cellreceptor alpha chain promoter, T cell receptor beta chain promoter, CD3gamma chain promoter, CD3 delta chain promoter, CD3 epsilon chainpromoter, and CD3 zeta chain promoter. In a specific embodiment, thepromoter is T cell receptor alpha chain promoter.

In another aspect, provided herein is a vector comprising anon-integrating gamma-retrovirus. In certain embodiments, thenon-integrating gamma-retrovirus comprises a mutated integrase. Incertain embodiments, the mutated integrase is mutated at a DDE motif. Incertain embodiments, the mutated integrase has a mutation selected fromthe group consisting of D124A, D124E, D124N, D124V, D183A, D183N, D124Aand D183A, D124A and D183N, D124E and D183A, D124E and D183N, D124N andD183A, D124N and D183N, D124V and D183A, and D124V and D183N.

In another aspect, provided herein is a T cell wherein a recombinantnucleic acid sequence encoding a chimeric antigen receptor (CAR) isintegrated at a first site within the genome of the cell such that theCAR is expressed by the cell at the surface of the cell, and whereinintegration of the nucleic acid encoding the CAR at the first sitereduces or prevents expression of a functional T cell receptor (TCR)complex at the surface of the cell. In certain embodiments, the nucleicacid sequence encoding the CAR is integrated at a single site within thegenome. In certain embodiments, the nucleic acid sequence encoding theCAR is integrated at two sites within the genome of the cell. In certainembodiments, the first site is an an exon of the gene encoding a proteinof the TCR complex.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, integration of the nucleic acidsequence encoding the CAR at the first site reduces or preventsexpression of a protein selected from the group consisting of T cellreceptor alpha chain, T cell receptor beta chain, CD3 gamma chain, CD3delta chain, CD3 epsilon chain, and CD3 zeta chain.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, expression of the integrated nucleicacid sequence in the T cell is under the control of an endogenouspromoter. In certain embodiments, the endogenous promoter is a T cellreceptor complex promoter. In certain embodiments, the endogenouspromoter is a promoter of a gene encoding a T cell receptor alpha chain,T cell receptor beta chain, CD3 gamma chain, CD3 delta chain, CD3epsilon chain, or CD3 zeta chain.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, the CAR binds to a cancer antigen.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, the T cell is selected from the groupconsisting of cytotoxic T lymphocyte (CTL), CD4+ subtype, CD8+ subtype,central memory T cell (TCM), stem memory T cell (TSCM), effector memoryT cell, effector T cell, Th1 cell, Th2 cell, Th9 cell, Th17 cell, Th22cell, Tfh (follicular helper) cell, and T regulatory cell.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, the T cell is derived from a human. Incertain embodiments, the T cell is a primary human T cell, a T cellderived from a CD34 hematopoietic stem cell, a T cell derived from anembryonic stem cell, or a T cell derived from an induced pluripotentstem cell.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, the nucleic acid sequence encoding theCAR is integrated into the first site by targeted homologousrecombination. In certain embodiments, the targeted homologousrecombination is carried out using a zinc-finger nuclease (ZFN), atranscription activator-like effector nuclease (TALEN), a clusteredregularly-interspersed short palindromic repeats (CRISPR) associatedprotein 9 (Cas9), Cpf1, Meganuclease or a Mega-Tal.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, the nucleic acid sequence encoding theCAR is integrated at a plurality of sites within the genome of the cell,and such that expression of the nucleic acid sequence encoding the CARat said plurality of sites is under the control of a differentendogenous promoter.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, the nucleic acid sequence encoding aCAR is also integrated at a second site within the genome of the cellsuch that the CAR is expressed by the cell at the surface of the cell.In certain embodiments, integration of the nucleic acid encoding the CARat the second site also reduces or prevents expression of a functionalTCR complex at the surface of the cell, wherein the first site and thesecond site are in different genes.

In certain embodiments of a T cell wherein a recombinant nucleic acidsequence encoding a CAR is integrated at a first site within the genomeof the cell, as described above, a second nucleic acid sequence encodinga second CAR is integrated at a second site within the genome of thecell such that the second CAR is expressed by the cell at the surface ofthe cell, and such that expression of the second nucleic acid sequenceis under the control of an endogenous promoter at the second site,wherein the first site and the second site are in different genes.

In another aspect, provided herein is a human T cell wherein apromotor-less recombinant nucleic acid sequence encoding a CAR isintegrated at a site in the genome of the cell, the site being the firstexon of the TCR alpha chain, such that the CAR is expressed undercontrol of the endogenous TCR alpha chain promoter, to produce the CARat the surface of the cell, and wherein integration of the CAR at thesite reduces or prevents expression of a functional TCR alpha chain. Incertain embodiments, the CAR binds to CD19.

In another aspect, provided herein is an isolated population of T cells,which comprises a plurality of the cell described above wherein arecombinant nucleic acid sequence encoding a CAR is integrated at afirst site within the genome of the cell, or a plurality of the celldescribed above that is a human T cell wherein a promotor-lessrecombinant nucleic acid sequence encoding a CAR is integrated at a sitein the genome of the cell.

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the cell describedabove wherein a recombinant nucleic acid sequence encoding a CAR isintegrated at a first site within the genome of the cell, or the celldescribed above that is a human T cell wherein a promotor-lessrecombinant nucleic acid sequence encoding a CAR is integrated at a sitein the genome of the cell; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of a population of Tcells, which population comprises a plurality of the cell describedabove wherein a recombinant nucleic acid sequence encoding a CAR isintegrated at a first site within the genome of the cell, or the celldescribed above that is a human T cell wherein a promotor-lessrecombinant nucleic acid sequence encoding a CAR is integrated at a sitein the genome of the cell; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating a subjectwith CAR therapy in need thereof, comprising administering to thesubject a therapeutically effective amount of the cell described abovewherein a recombinant nucleic acid sequence encoding a CAR is integratedat a first site within the genome of the cell, or the cell describedabove that is a human T cell wherein a promotor-less recombinant nucleicacid sequence encoding a CAR is integrated at a site in the genome ofthe cell.

In another aspect, provided herein is a method of treating a subjectwith CAR therapy in need thereof, comprising administering to thesubject a pharmaceutical composition described above comprising c atherapeutically effective amount of the cell described above wherein arecombinant nucleic acid sequence encoding a CAR is integrated at afirst site within the genome of the cell, or the cell described abovethat is a human T cell wherein a promotor-less recombinant nucleic acidsequence encoding a CAR is integrated at a site in the genome of thecell.

In another aspect, provided herein is a method of treating a subjectwith CAR therapy in need thereof, comprising administering to thesubject a therapeutically effective amount of the cell populationdescribed above comprising a plurality of the cell described abovewherein a recombinant nucleic acid sequence encoding a CAR is integratedat a first site within the genome of the cell, or a plurality of thecell described above that is a human T cell wherein a promotor-lessrecombinant nucleic acid sequence encoding a CAR is integrated at a sitein the genome of the cell.

In another aspect, provided herein is a method of treating a subjectwith CAR therapy in need thereof, comprising administering to thesubject a pharmaceutical composition described above comprising atherapeutically effective amount of a population of T cells, whichpopulation comprises a plurality of the cell described above wherein arecombinant nucleic acid sequence encoding a CAR is integrated at afirst site within the genome of the cell, or the cell described abovethat is a human T cell wherein a promotor-less recombinant nucleic acidsequence encoding a CAR is integrated at a site in the genome of thecell.

In certain embodiments of the methods described above for treating asubject with CAR therapy in need thereof, the subject has cancer, andthe CAR binds to a cancer antigen of the cancer. In a specificembodiment, the cancer is leukemia.

In certain embodiments of the methods described above for treating asubject with CAR therapy in need thereof, the subject has a tumor.

In certain embodiments of the methods described above for treating asubject with CAR therapy in need thereof, the subject is a human, andthe cell is derived from a human.

In certain embodiments of the methods described above for treating asubject with CAR therapy in need thereof, the cell is autologous to thesubject. In certain embodiments of the methods described above fortreating a subject with CAR therapy in need thereof, the cell isnon-autologous to the subject.

In another aspect, provided herein is a method of generating a T cellthat expresses a chimeric antigen receptor (CAR) and lacks a functionalT cell receptor (TCR) complex, comprising: introducing into a T cell:(i) a nucleic acid sequence encoding a CAR, and (ii) a homologousrecombination system suitable for targeted integration of the nucleicacid sequence at a site within the genome of the cell, whereby thehomologous recombination system integrates the nucleic acid sequenceencoding the CAR at the site within the genome of the cell such thatintegration of the CAR at the site reduces or prevents expression of afunctional T cell receptor complex at the surface of the cell, therebygenerating a T cell that expresses the CAR and lacks a functional TCRcomplex.

In certain embodiments of a method of generating a T cell that expressesa CAR and lacks a functional TCR complex, as described above, expressionof the CAR is under the control of an endogenous promoter. In certainembodiments, the endogenous promoter is a promoter of a gene encoding aT cell receptor alpha chain, T cell receptor beta chain, CD3 gammachain, CD3 delta chain, CD3 epsilon chain, or CD3 zeta chain.

In certain embodiments of a method of generating a T cell that expressesa CAR and lacks a functional TCR complex, as described above, thehomologous recombination system comprises a zinc-finger nuclease (ZFN),a transcription activator-like effector nuclease (TALEN), or clusteredregularly-interspersed short palindromic repeats (CRISPR) associatedprotein 9 (Cas9), Cpf1, Meganuclease or a Mega-Tal.

In certain embodiments of a method of generating a T cell that expressesa CAR and lacks a functional TCR complex, as described above, thenucleic acid sequence encoding the CAR that is introduced into the cellis contained in a targeting construct. In certain embodiments, thetargeting construct comprises adeno-associated virus 2 (AAV2) sequences.In certain embodiments, the targeting construct is packaged into anatural or recombinant adeno-associated virus (AVV) viral particle. Incertain embodiments, the AAV particle comprises AAV6 sequences.

In certain embodiments of a method of generating a T cell that expressesa CAR and lacks a functional TCR complex, as described above, thenucleic acid sequences encoding the CAR in the targeting construct arenot operably linked to a promoter.

In certain embodiments of a method of generating a T cell that expressesa CAR and lacks a functional TCR complex, as described above, thetargeting construct comprises in 5′ to 3′ order: a first viral sequence,a left homology arm, a nucleic acid sequence encoding a self-cleavingporcine teschovirus 2A, the nucleic acid sequence encoding the CAR, apolyadenylation sequence, a right homology arm, and a second viralsequence. In certain embodiments, the first or the second viral sequenceis from an adeno-associated virus (AAV). In certain embodiments, the AAVis AAV2, AAV5 or AAV6.

In another aspect, provided herein is an induced pluripotent stem cell,wherein a recombinant nucleic acid sequence encoding a chimeric antigenreceptor (CAR) is integrated at a first site within the genome of thecell such that the CAR is expressed by the cell at the surface of thecell, and wherein integration of the nucleic acid encoding the CAR atthe first site reduces or prevents expression of a functional T cellreceptor (TCR) complex at the surface of the cell.

6. DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show analysis of targeted integration of a CAR into the TCRalpha constant (TRAC) locus. FIG. 1A shows a schematic of tailorednuclease (TALEN or CRISPR/Cas9)-induced targeted integration into TCRalpha constant (TRAC) locus. The targeting construct (AAV6) contains theCAR gene flanked by homology sequences (LHA and RHA). Once integratedCAR expression is driven by the endogenous TCRa promoter while the TRAClocus is disrupted. TRAV: TCR alpha variable region. TRAJ: TCR alphajoining region. 2A: the self cleaving Porcine teschovirus 2A sequence.pA: bovine growth hormone PolyA sequence. FIG. 1B shows representativeTCR/CAR flow plot 5 days after transfection of T cells with TRAC TALENmRNA and addition of AAV6 at the noted MOI (multiplicity of infection).FIG. 1C shows a bar-graph of the percentage of TCR disruption (KO:knockout) and targeted integration (KI: knockin) depending on the AAV6MOI. Percentages were assessed by FACS analysis. FIG. 1D shows averageCAR expression mean fluorescence intensity (MFI) 5 days after CARvectorization (choosing an adapted vector for expressing the CAR,integration of the CAR coding into the cell) into T cells (n=6 to 8independent experiments). FIG. 1E shows coefficient of variation of theCAR+ T cells measuring the dispersion in the CAR expression (ratio ofthe standard deviation to the mean). TRAC-P2A-1928z: Targetedintegration into TRAC. SFG-1928z: semi-random integration using the SFGretrovirus. ****P<0.0001 (unpaired T-test).

FIGS. 2A-2E show analysis of targeted integration of a CAR into the TCRalpha constant (TRAC) locus. FIG. 2A shows flow cytometry analysisshowing CAR and TCR expression. TRAC-P2A-1928z were generated as in FIG.1; TALEN-generated TCR− cells were transduced with SFG-1928z retrovirus;and TCR+ cells were transduced with either SFG-1928z or SGF-P28zretrovirus. FIG. 2B shows cumulative cell counts of indicated CAR Tcells upon weekly stimulation with CD19+ target cells. Arrows indicatestimulation time points. FIG. 2C shows cytotoxic activity using an 18 hrbioluminescence assay, using firefly luciferase (FFL)-expressing NALM6as targets cells. Data are means±SD. FIG. 2D and 2E show FFL-NALM6bearing mice, which were treated with 2×10⁵ CAR T cells. Tumor burdenshown as bioluminescent signal quantified per animal every week over a40-day period. Quantification is the average photon count of ventral anddorsal acquisitions per animal at all given time points, and it isexpressed as radiance. Each line represents one mouse. n=7 mice pergroup. The lower right figure is Kaplan-Meier analysis of survival ofmice in FIG. 2D and 2E.

FIGS. 3A-3J show that TRAC-CAR T cells outperform conventional CAR Tcells by preventing exhaustion in vivo. FIG. 3A showsCRISPR/Cas9-targeted CAR gene integration into the TRAC locus. Top, TRAClocus; middle, rAAV6 containing the CAR cassette flanked by homologyarms; bottom, edited TRAC locus. FIG. 3B shows representative TCR/CARflow plots 4 days after TRAC targeting. FIGS. 3C and 3D show CAR meanfluorescence intensity (MFI) (FIG. 3C) and CAR MFI coefficient ofvariance (FIG. 3D) of CAR+ T cells (n=12 independent experiments, 6donors). FIG. 3E shows Kaplan-Meier analysis of survival of mice. FIGS.3F-3J show NALM-6-bearing mice were treated with 1×10⁵ CAR T cells (n=7per group; dot=one mouse), and euthanized at days 10 and 17 afterinfusion; bone marrow CAR T cells and NALM-6 cells were analysed andcounted by FACS (TRAC-1928z, circles; RV-1928z-TCR—, squares; RV-1928z,triangles). FIG. 3F shows CAR T cells. FIG. 3G shows tumour (GFP+CD19+)cells. FIG. 3H shows CAR T cells to tumour ratio. FIG. 31 showspercentage of effector memory (CD62L−CD45RA−) and effector(CD62L−CD45RA+) in CAR T cells at day 17. FIG. 3J shows percentage ofCAR T cells expressing exhaustion markers; quantified by FACS at day 17(inhibitory receptor expression shown from inner to outer rings TIM3,LAG3 and PD1, respectively). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001(Welch's two samples t-test (FIGS. 3C and 3D); log-rank Mantel-Cox test(FIG. 3E); Mann-Whitney (FIGS. 3F-3I)). All data are means±s.d. See alsoFIGS. 7-10.

FIGS. 4A-4E show that TRAC-CAR T cells display reduced constitutivesignalling and antigen-induced differentiation. FIG. 4A shows FACSanalysis of activation, memory and exhaustion markers in T cells (day 5after infusion; representative of 3 donors; pie chart for CD62L/D45RAexpression (n=3, 3 donors). FIG. 4B shows CAR expression and CD3ζ ITAMphosphorylation (representative of 3 donors). RV-19Del, retrovirallyexpressed CD19-specific CAR lacking signalling domains. FIG. 4C showsphospho-CD3t MFI in the CAR+ population (n=3, 3 donors; **P<0.05Mann-Whitney test). FIG. 4D shows CD62L/CD45RA expression in CAR T cellsstimulated 1, 2 or 4 times. Pie charts show the phenotypes of the CAR+ Tcells (n=3-5 on different donors) (A, CD45RA+ CD62L+; B, CD45RA− CD62L+;C, CD45RA− CD62L−; D, CD45RA+ CD62L−). FIG. 4E shows heat map of T-bet,EOMES and GATA3 expression in CAR T cells collected as in FIG. 4D;fold-increase value of 1 represents to TRAC-1928z, 1 stimulation (n=2, 2donors). All data are means±s.d. See also FIG. 12.

FIGS. 5A-5G show that the endogenous TRAC promoter surpasses otherlocus/promoter combinations in vivo. FIG. 5A shows a schematic ofCRISPR/Cas9-targeted promoter-CAR integration into the TRAC locus. Top:TRAC locus; bottom: rAAV6 containing the promoter-CAR-polyA cassetteflanked by homology arms. FIG. 5B shows a schematic ofCRISPR/Cas9-targeted promoter-less CAR integration into the B2M locus.Top: B2M locus; bottom: rAAV6 containing a promoter-less CAR cassetteflanked by homology arms. FIG. 5C shows representative B2M/CAR orTCR/CAR flow plots 4 days after vectorization of T cells. FIG. 5D showsCAR mean fluorescence intensity (MFI) at day 4 (n=4-7 independentexperiments; 4 donors) (TRAC-LTR-1928z, B2M-1928z, TRAC-1928z andTRAC-EF1α-1928z, left to right, respectively). FIG. 5E shows CARexpression. Left panel: CAR expression (representative histogram) at day4. Right: FACS analysis of activation, memory, and exhaustion markers ofCAR T cells at day 5 (representative of 3 donors). FIG. 5F shows CAR Tcells stimulated on CD19+ target cells 0, 1, 2 or 4 times. Pie chartsshow the CD62L/CD45RA phenotypes of CAR+ T cells (n=3-5 independentexperiments on different donors) (A, CD45RA+ CD62L+; B, CD45RA− CD62L+;C, CD45RA− CD62L−; D, CD45RA+ CD62L−). FIG. 5G shows tumour burden(average radiance) of NALM-6-bearing mice treated with 1×10⁵ CAR T cells(n=6; line=one mouse). Tumour burden was quantified weekly over a 50-dayperiod using bioluminescence imaging (BLI). Quantification is theaverage photon count of ventral and dorsal acquisitions per animal atall given time points. Each line represents one mouse. FIG. 5H showsKaplan-Meier analysis of the mice survival. (A): TRAC-EF1a-1928z CAR Tcells, (B): B2M-1928z CAR T cells, (C): TRAC-LTR-1928z CAR T cells, (D):TRAC-1928z CART cells, TRAC-1928z labeled as indicated. **P<0.01,***P<0.001, ****P<0.0001 (Welch's two samples t-test for FIG. 5D);Log-rank Mantel-Cox test for FIG. 5G; Mann-Whitney test for FIG. 5H).All data are means±SD. See also FIGS. 14 and 15.

FIGS. 6A-6F show that the TRAC locus affords optimal regulation ofcell-surface CAR expression. FIG. 6A shows a representative histogram ofCAR expression before and after co-culture with CD19+ target cells. FIG.6B shows CRISPR/Cas9-targeted integration of a CAR-GFP fusion gene intoTRAC locus. FIG. 6C: Upper, LNGFR/CAR expression of the bicistronicCAR-P2A-LNGFR CAR T cells before and after co-culture with CD19+ targetcells. Lower, GFP/CAR expression of CAR-GFP fusion targeted into theTRAC locus or randomly integrated with the RV vector (representative of3 independent experiments on 3 donors). FIG. 6D shows CAR expression.Left panel: representative histogram of the CAR expression 5 dayspost-vectorization. Right panel: relative CAR MFI (1=MFI at 0 h) of CARTcells after 1, 2 or 4 stimulations (indicated by arrows; n=3-7independent experiments on different donors). FIG. 6E shows relative CARRNA levels (1=TRAC RNA level) 5 days post-vectorization. FIG. 6F shows atime course analysis of CAR RNA levels (1=RNA level at Oh) in CAR Tcells stimulated once on CD19+ target cells (n=3 independent experimentson 3 donors; CAR T cells as in FIG. 6D, top to bottom). All data aremeans±SD. *P<0.05, **P<0.01, ***P<0.001 (ANOVA F-test with Bonferronicorrection (FIG. 6D), and Mann-Whitney test (FIG. 6E)). See also FIG.16. The lower line represents the CAR surface levels (FIG. 6D) or CARRNA levels (FIG. 6F) in TRAC-1928z CAR T cells.

FIGS. 7A-7G show CRISPR/Cas9-mediated CAR gene targeting into the TRAClocus. FIG. 7A, Top, TRAC locus (SEQ ID NO:41) with the 5′ end (grey) ofthe TRAC first exon, the TRAC gRNA (TGT . . . GAC, lower strand) and thecorresponding PAM sequence (GGG, immediately left of TGT . . . GAC). Thetwo arrows indicate the predicted Cas9 double strand break. Bottom,CRISPR/Cas9-targeted integration into the TRAC locus. The targetingconstruct (AAV) contains a splice acceptor (SA), followed by a P2Acoding sequence, the 1928z CAR gene and a polyA sequence, flanked bysequences homologous to the TRAC locus (LHA and RHA, left and righthomology arm). Once integrated, the endogenous TCRa promoter drives CARexpression, while the TRAC locus is disrupted. TRAY, TCRa variableregion; TRAJ, TCRa joining region; 2A, the self-cleaving Porcineteschovirus 2A sequence. pA: bovine growth hormone polyA sequence. FIG.7B shows a timeline of the CAR targeting into primary T cells. FIG. 7Cshows representative TCR/CAR flow plots 4 days after transfection of Tcells with Cas9 mRNA and TRAC gRNA and addition of AAV6 at the indicatedmultiplicity of infection. FIG. 7D shows percentage of TCR disruption 4days post transfection of the Cas9 mRNA and the TRAC gRNA measured byFACS analysis of the TCR expression (n=5). FIG. 7E shows showspercentage of knock-in depending on the AAV6 multiplicity of infectionmeasured by FACS analysis of the CAR expression (n=4). FIG. 7F showspercentage of CAR+ cells in the TCR-negative population (n=4). FIG. 7Gshows percentage of TCR-positive (lower bar) and TCR-negative (upperbar) in the CAR+ population analysed by FACS (n=4).

FIGS. 8A-8E show whole-genome mapping of the AAV6 TRAC-1928z integrationusing the TLA technology. FIG. 8A shows a schematic representation ofthe TLA technology (de Vree et al., Nat. Biotechnol. 32:1019-1025(2014)). For this study, two sets of primers targeting the CAR and theleft homology arm have been used. FIG. 8B shows TCR/CAR FACS plot of theTRAC-1928z CAR T cells used for the TLA analysis. CAR T cells have beenprocessed as in FIG. 7B and expanded for 2 weeks. FIG. 8C shows TLAsequence coverage across the human genome using 1928z CAR specificprimers (CD28-specific forward: 5′-ACAATGAGAAGAGCAATGGA-3′ (SEQ IDNO:39) and scFV-specific reverse: 5′-GAGATTGTCCTGGTTTCTGT-3′ (SEQ IDNO:40)). The chromosomes are indicated on the y axis, the chromosomalposition on the x axis. TRAC-encoded CAR T cells were produced as inFIG. 3 and expanded for 10 days before processed for analysis. Theprimer set was used in an individual TLA amplification. PCR productswere purified and library prepped using the Illumina NexteraXT™ protocoland sequenced on an Illumina Miseg™ sequencer. Reads were mapped usingBWA-SW, which is a Smith-Waterman alignment tool. This allows partialmapping, which is optimally suited for identifying break-spanning reads.The human genome version hg19 was used for mapping. FIG. 8D shows TLAsequence coverage aligned on the AAV-TRAC-1928z sequence (Targetingsequence flanked by ITRs). The grey vertical bars on top represent thecoverage at the shown positions. The coverage showed integration of theAAV ITRs in fraction of reads. The coverage comparison between ITR andCAR integration at the 5′ and 3′ ends of the TRAC homology arms locusallow the measurement of faithful and unfaithful homologousrecombination shown in FIG. 8E. FIG. 8E shows final results from the TLAanalysis.

FIGS. 9A-9C show in vitro cytotoxicity activity and proliferationresponse of TRAC-CAR T cells. FIG. 9A shows representative flowcytometry analysis showing CAR and TCR expression. TRAC-1928z CAR Tcells were generated as in FIG. 3B; CRISPR/Cas9-generated TCR− T cellswere transduced with RV-1928z retroviral vector; TCR+ cells weretransduced with either RV-1928z or RV-P28z (PSMA-specific CAR).TCR-negative T-cell purification was performed using magnetic beads oncolumn. FIG. 9B shows cytotoxic activity using an 18 h bioluminescenceassay, using firefly luciferase (FFL)-expressing NALM-6 as targets cells(n=3 independent experiments on 3 healthy donors). FIG. 9C showsrepresentative cumulative cell counts of CAR T cells upon weeklystimulation with CD19+ target cells. Arrows indicate stimulation timepoints (n=3 independent experiments on 3 healthy donors).

FIGS. 10A-10I show that TRAC-CAR T cells outperform conventional CAR Tcells in vivo. FIG. 10A shows NALM-6-bearing mice were treated with2×10⁵ (left), 1×10⁵ (middle) or 5×10⁴ (right) CAR T cells. Tumour burdenwas quantified weekly over a 100-day period using BLI. Quantification isthe average photon count of ventral and dorsal acquisitions per animalat all given time points. Each line represents one mouse. Some groupsare pooled from two to three independent experiments from differenthealthy donors, representing n=6-20 mice per group. Lower, Kaplan-Meieranalysis of survival of mice. FIGS. 10B-10F show NALM-6-bearing micewere treated with 1×10⁵ indicated CAR T cells. At 10 and 17 days afterCAR T-cell infusion, 7 mice per group were euthanized and bone marrowcells were collected. CAR T cells and NALM-6 cells were analysed andcounted with flow cytometry. FIG. 10B shows representative FACS analysisof tumour cells (CD19+GFP+) in the bone marrow at day 17. FIG. 10C showsrepresentative FACS analysis of exhaustion markers PD1 and TIM3 in bonemarrow CAR T cells at day 17. FIG. 10D show representative FACS analysisof exhaustion markers PD1 and LAG3 in bone marrow CART cells at day 17.FIG. 10E shows CAR MFI of the CAR+ cells in the bone marrow (each dotrepresents one mouse). FIG. 10F shows coefficient of variation measuringthe dispersion in the CAR expression of the CAR+ population (ratio ofthe standard deviation to the mean; each dot represents one mouse). FIG.10G shows that RV-1928z CAR design allows the co-expression of the CARand LNGFR from the same LTR promoter by using a self-cleaving P2Asequence. LTR, long terminal repeat, SD, splice donor site; SA, spliceacceptor site; 2A, Porcine teschovirus self-cleaving 2A sequence. FIG.10H shows representative flow cytometry plots of RV-1928z transduced Tcells cultured in vitro or in vivo (extracted from bone marrow) andlabelled to detect CAR and LNGFR expression. FIG. 10I shows a comparisonbetween CAR MFI in the RV-1928z T cells and the tumour burden (NALM-6count) in the bone marrow.

FIGS. 11A-11J show that TRAC-19BBz CAR T cells outperform conventional19BBz CAR T cells by preventing exhaustion in vivo. FIGS. 11A and 11Bshow results compiled from the average CAR MFI (FIG. 11A) andcoefficient of variation (FIG. 11B) of CAR+ T cells obtained from threeindependent transfections or transductions. The T cells used for thesethree experiments have been isolated from blood of three differenthealthy donors. FIG. 11C: Left, activation, memory, and exhaustionmarkers of CAR T cells analysed by flow cytometry 5 days after genetransfer. Right, plots indicate the phenotypes of the CAR+ T cellsmeasured by flow cytometry analysis of CD62L and CD45RA expression 5days after CAR vectorization; A: CD45RA+CD62L+; B: CD45RA− CD62L+; C:CD45RA− CD62L−; D: CD45RA+ CD62L−. FIG. 11D shows relative CAR MFI(1=MFI at 0 h) after CAR T cells being activated 1, 2 or 4 times onCD19+ target cells over a 48 h periods (n=3 independent experiments,arrows indicate stimulation time points) (TRAC-19bbz lower line,RV-19bbz upper line). FIG. 11E shows CAR T cells stimulated on CD19+target cells either 1, 2 or 4 times in 48 h period were analysed by flowcytometry. Plots indicate the phenotypes of the CAR+ T cells measured byflow cytometry analysis of CD62L and CD45RA expression (averageproportion from 3 independent experiments). FIG. 11F showsFFL-NALM-6-bearing mice were treated with 1×10⁵ CAR T cells. Tumourburden shown as bioluminescent signal quantified per animal every weekover a 21-day period. n=6 mice per group. FIGS. 11G-11J showNALM-6-bearing mice were treated with 1×10⁵ CAR T cells. At 10 and 17days after CAR T-cell infusion, 7 mice per group were euthanized andbone marrow cells were collected. CAR T cells and NALM-6 cells wereanalysed and counted with flow cytometry. Each dot represents one mouse.FIG. 11 G shows CAR T cells count in marrow (n=7). FIG. 11H shows tumour(CD19+GFP+ NALM-6) cells count in bone marrow (n=7). FIG. 11I showseffector/tumour ratio in the bone marrow (n=7). FIG. 11J showsexhaustion marker analysis from bone marrow T cells collected at day 17and analysed by flow cytometry (inhibitory receptor expression shownfrom inner to outer rings TIM3, LAG3 and PD1, respectively). Representedas the average percentage of cells expressing the indicated markers(n=7). *P<0.05, **P<0.01, ***P<0.001 (Mann-Whitney test (FIGS. 11A and11B) ANOVA F-test (FIG. 11D).

FIGS. 12A-12D show that TRAC-CAR T cells show reduced tonic signallingand antigen-induced differentiation in vitro. FIG. 12A showsrepresentative FACS analysis of T cells differentiation markers 5 daysafter the CAR gene transfer. FIG. 12B shows representative FACS analysisof the CAR T cell differentiation markers after 1, 2 or 4 stimulationson CD19+ target cells. FIG. 12C shows CAR T cells expansion whenstimulated 1, 2 or 4 times on CD19+ target cells over a 48 h period. Nonoticeable difference in the proliferation was found between the three1928z CAR T cells conditions. FIG. 12D shows percentage of CAR T cellswith positive expression of IFNγ, TNFα or IL-2 after intracellularstaining at the end of the protocol in FIG. 4D (n=2 independentexperiments on 2 donors) (groups of bars left to right TRAC-1928z,RV1928z and RV-P28z, respectively).

FIGS. 13A-13F show that TRAC-CAR T cells show delayed in vitroantigen-induced differentiation compared to lowly or highly transducedRV-CAR T cells. FIG. 13A shows a representative histogram of the CARexpression 5 days after transduction of different volumes of retroviralsupernatant in μl (representative of 3 independent experiments; totaltransduction volume 2 ml). FIG. 13B shows percentage of CAR+ T cells asa function of the volume of retroviral supernatant analysed by FACS 5days after transduction (n=3 donors). FIG. 13C shows CAR meanfluorescence intensity (MFI) of T cells as a function of the volume ofretroviral supernatant analysed by FACS 5 days after transduction (n=3donors). FIG. 13D shows CAR coefficient of variation as a function ofthe volume of retroviral supernatant analysed by FACS 5 days aftertransduction (n=3 donors). FIG. 13E shows average CAR MFI of CAR T cells5 days after transduction (n=3 donors). High=1,000 μl, and low=30 μl.FIG. 13F shows CAR T cells stimulated on CD19+ target cells either 1, 2or 4 times in 48 h period were analysed by flow cytometry. Plotsindicate the phenotypes of the CAR-positive T cells measured by flowcytometry analysis of CD62L and CD45RA expression (average proportionfrom of 3 independent experiments) (A, CD45RA+ CD62L+; B, CD45RA−CD62L+; C, CD45RA− CD62L−; D, CD45RA+ CD62L−).

FIGS. 14A-14F CAR gene expression using different promoters at distinctloci influences tonic signalling levels in vitro. FIG. 14A shows adiagram of CRISPR/Cas9-targeted integration into the TRAC locus. Thetargeting construct (AAV) contains a splice acceptor (SA), followed by aP2A coding sequence, the 1928z CAR gene and a polyA sequence, flanked bysequences homologous to the TRAC locus (LHA and RHA: left and righthomology arm). Once integrated, the endogenous TCRa promoter drives CARexpression, while TRAC locus is disrupted. TRAV: TCR alpha variableregion. TRAJ: TCR alpha joining region 2A: the self-cleaving Porcineteschovirus 2A sequence. FIG. 14B shows a diagram ofCRISPR/Cas9-targeted promoter integration into the TRAC locus. Thetargeting construct (AAV) contains the 1928z CAR coding sequence in thereverse orientation, under the control of an exogenous promoter, thelong version of the human elongation factor 1 alpha promoter (EF1α), theenhancer sequence from the gamma retrovirus used in FIGS. 3 and 4(Mo-MLV LTR here called LTR) or the phosphoglycerate kinase (PGK)promoter and a polyA sequence, flanked by sequences homologous to theTRAC locus (LHA and RHA: left and right homology arm). TRAV: TCR alphavariable region. TRAJ: TCR alpha joining region. FIG. 14C shows aschematic of tailored CRISPR/Cas9-induced targeted integration into theB₂M locus. The targeting construct (AAV) contains the CAR gene flankedby homology sequences (LHA and RHA). Once integrated, the endogenous B₂Mpromoter drives CAR expression. FIG. 14D shows a schematic ofCRISPR/Cas9-targeted promoter integration into the B₂M locus. Thetargeting construct (AAV) contains the 1928z CAR gene in the reverseorientation, under the control of an exogenous promoter, the humanelongation factor 1 alpha promoter (EF1α), the phosphoglycerate kinase(PGK) promoter or a truncated version of the PGK (PGK100) and a polyAsequence, flanked by sequences homologous to the B₂M locus (LHA and RHA:left and right homology arm). FIG. 14E shows average CAR meanfluorescence intensity (MFI) analysed by FACS 4 days after transduction(n=3 to 7 independent experiments and 4 different donors). pA: bovinegrowth hormone polyA sequence for all targeting constructs. FIG. 14Fshows analysis of CAR T cells 5 days after vectorization. Left panel:representative histogram of the CAR expression 5 days after itsvectorization into T cells. Middle panel: Activation, memory, andexhaustion markers of CAR T cells analysed by flow cytometry 5 daysafter the vectorization of the CAR. Right panel: Plots indicate thephenotypes of the CAR positive T cells measured by flow cytometryanalysis of CD62L and CD45RA expression 5 days after vectorization ofthe CAR (A, CD45RA+ CD62L+; B, CD45RA− CD62L+; C, CD45RA− CD62L−; D,CD45RA+ CD62L−).

FIGS. 15A-15G show that CAR gene expression using different promoters atdistinct loci influences antigen-induced differentiation and exhaustionin vivo. FIG. 15A shows representative FACS analysis of the CAR T-celldifferentiation markers after 1, 2 or 4 stimulations on CD19+ targetcells. FIG. 15B shows CAR T-cell expansion when stimulated 1, 2 or 4times on CD19+ target cells over a 48 h period (groups of dots left toright TRAC-LTR-1928z, B2M-1928z, TRAC-1928z and TRAC-EF1a-1928z,respectively). No apparent difference in the proliferation was foundbetween the four 1928z CAR T cells conditions. FIGS. 15C-15E showNALM-6-bearing mice were treated with 1×10⁵ CAR T cells. At 10 and 17days after CAR T cell infusion, 7 mice per group were euthanized andbone marrow cells were collected. CAR T cells and NALM-6 cells wereanalysed and counted with flow cytometry. Each dot represents one mouse.FIG. 15F shows percentage of effector memory (‘Eff mem’, CD62L−CD45RA−)and effector (‘Eff’, CD62L−CD45RA+) in the bone marrow CAR T cells atday 17 (n=7 mice). FIG. 15G shows exhaustion marker analysis from bonemarrow T cells collected at day 17 and analysed by flow cytometry.Represented as the average percentage of cells expressing the indicatedmarkers (n=7 mice) (inhibitory receptor expression shown from inner toouter rings TIM3, LAG3 and PD1, respectively).

FIGS. 16A-16B show that locus-promoter configuration controls CARprotein expression and transcriptional response upon CAR T cellactivation. FIG. 16A: Left panel: representative histogram of the CARexpression 5 days after its vectorization into T cells. Right panel:relative CAR MFI (1=MFI at Oh) after CAR T cells being activated 1, 2 or4 times on CD19+ target cells over a 48 h period. FIG. 16B shows acomparison between CAR MFI and CAR RNA relative level before stimulation(n=3 independent experiments on 3 donors). The lower line represents theCAR surface levels in TRAC-1928z CAR T cells.

FIG. 17 shows gene-expression profiles associated with the activationand memory formation of CD8+ T cells. Genes upregulated (Up) ordownregulated (Down) in infection-exposed OT-I cells relative to theirexpression in naive OT-I cells was quantified at various time pointsduring infection. Ten clusters with the most dynamic expression byK-means clustering analysis are shown, with a change in expression ofover 1.4-fold. Each line represents a single probe; numbers in bottomright corners indicate number of probes; above plots, genes of interestin each cluster (taken from Best et al., Nature Immunol. 14:404-413(2013)).

FIG. 18 shows Moloney murine leukemia virus (MLV) amino acid sequence ofintegrase wild type (SEQ ID NO:1) and mutants D124A (SEQ ID NO:2), D124E(SEQ ID NO:3), D124N (SEQ ID NO:4), D124V (SEQ ID NO:5), D183A (SEQ IDNO:6), D183N (SEQ ID NO:7), D124A and D183A (SEQ ID NO:8), D124A andD183N (SEQ ID NO:9), D124E and D183A (SEQ ID NO:10), D124E and D183N(SEQ ID NO:11), D124N and D183A (SEQ ID NO:12), D124N and D183N (SEQ IDNO:13), D124V and D183A (SEQ ID NO:14), and D124V and D183N (SEQ IDNO:15).

Where reference is made in the description of the drawings to color, thedrawings have been converted to grayscale.

7. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunotherapy and specifically totargeted cell therapies based on genetically engineering T cells toexpress a therapeutic transgene under desired conditions. Describedherein is a method for generating T cells for immunotherapy by targetingthe integration of a therapeutic transgene into the genome of a T cellsuch that the transgene is placed under control of an endogenouspromoter. It will be understood that reference to a transgene (in thesingular) as described herein applies also to one or more transgenes (inthe plural) unless context indicates otherwise. The invention provides astrategy for T cell therapy that utilizes genome editing to place one orseveral therapeutic transgenes under the control of one or moreendogenous promoters to provide controlled spatio-temporal expression intherapeutic T cells. The invention provides for a T cell to beengineered to express a therapeutic transgene, or a variety oftherapeutic transgenes, where expression of the transgene can be madedependent on the location of the T cell (e.g., expression of a transgeneonly in proximity to a tumor), or at defined time points (e.g., beforeor after engaging a tumor cell) by use of endogenous promoters thatprovide for expression accordingly. The cells and methods of theinvention can thus be used to increase the efficacy and safety oftherapeutic T cells.

The invention relates to placing a therapeutic transgene under controlof an endogenous promoter to achieve a desired transgene expressionprofile in the T cell. An endogenous promoter is selected so as toregulate the expression characteristics of the transgene, for example,the timing of transgene expression and/or the level of transgeneexpression. Regulating expression of the transgene by placing it undercontrol of an endogenous promoter eliminates the need for administeringsmall molecule drugs to induce expression of a transgene, immunogeniccomponents, and viral vectors encoding internal promoters andtransgenes. By utilizing endogenous promoters, the T cells areengineered to autonomously regulate expression of transgenes such thattransgene expression, for example, where and when transgene expressionis activated, preferably occurs in a defined program that relies on thecoordinated endogenous response of the T cell to environmental cues(e.g., proximity to a target antigen, cytokine, and/or costimulatoryligand). Thus, in a specific embodiment, the T cell is engineered suchthat an endogenous promoter is used that responds to microenvironmentalcues, resulting in spatially and temporally predictable transgeneexpression governed by the endogenous promoter.

In a specific embodiment, the therapeutic transgene encodes atherapeutic protein. In another specific embodiment, the therapeutictransgene encodes a therapeutic RNA.

In a preferred embodiment, the present invention relates toimmunotherapy and specifically to targeted cell therapies based on thegenetic replacement of a component of the T cell receptor (TCR) complexwith sequences encoding a CAR that reprograms T cell specificity andfunction. As disclosed by way of example herein, gene editing wasutilized to generate histocompatible T cell products with stable andhomogeneous CAR expression. In addition, the gene editing approachresults in the disruption of the targeted gene encoding a component ofthe TCR complex, which enhances the function of the CAR-T cells byreducing graft versus host reactivity that would have been mediated bythe TCR complex. It also can be used for patient auto-immune diseases,usually not included in the clinical Trial. Inactivating their TCR canbe used to improve safety for these patients.

In a specific embodiment, described herein is a method for a one-stepgeneration of universal CAR T cells by targeting the integration of aCAR gene cassette, preferably promoter-less, into a gene encoding apolypeptide required for functional expression of a T cell receptor(TCR) complex. The term “universal” denotes that the T cells are notlimited to autologous use, but can also be used non-autologously. In oneembodiment, this approach can take advantage of the regulated expressionof a component of the TCR complex to drive the expression of the CAR inthe cell. In addition, the integration of the CAR cassette disrupts orreduces the expression of a polypeptide required for a functional TCRcomplex, for example, by preventing the proper assembly of the TCRcomplex at the cell surface, leading to TCR negative cells. The methodis suitable with the commonly used genome editing platforms, such aszinc-finger nuclease (ZFN), transcription activator-like effectornuclease (TALEN), and clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, Meganuclease or aMega-Tal, and results in homologous recombination at a target site inthe genome of the cell. As disclosed herein, conditions were establishedyielding up to 50% of universal CAR T cells, combining target genedisruption and CAR targeted insertion in a single step. The resultsdisclosed herein shows that the methods utilizing an endogenous TCRpromoter provided the benefits of single integration as well asconsistent and predictable expression. In addition, the method providedunexpected benefits of improved T cell function and persistence. Mostimportantly, T cells expressing the CAR from the TCR locus exhibitedhigher in vitro and in vivo tumor lysis activity, increasedproliferation and persistence than retrovirally-transduced CAR T cells,while removing their Graft versus host disease potential. Moreover, thisnew methodology opens the possibility of generating autologous CAR Tcells for patients suffering from autoimmune disorders. The methodsdescribed herein, which combine the scalability of universal T cellmanufacturing with the uniformity and safety of targeted CAR geneintegration, are useful for CAR therapy and for the development ofoff-the-shelf CAR therapy.

7.1 T Cells

In one embodiment, the invention provides a T cell, wherein atherapeutic transgene is integrated at a site within the genome of thecell such that expression of the transgene is under control of anendogenous promoter of the T cell. In a preferred embodiment, theinvention provides a T cell, wherein a recombinant nucleic acid sequenceencoding a chimeric antigen receptor (CAR) is integrated at a sitewithin the genome of the cell such that the CAR is expressed by the cellat the surface of the cell, and wherein integration of the nucleic acidencoding the CAR at the site reduces or prevents expression of afunctional T cell receptor (TCR) complex at the surface of the cell. Ina preferred embodiment, the recombinant cells can be used to enhance orprovide an immune response against a desired target. In anotherembodiment, the recombinant cells can be used to inhibit an undesirableimmune response. Preferably, the cells are derived from a human (are ofhuman origin prior to being made recombinant) (and human-derived cellsare particularly preferred for administration to a human in the methodsof treatment of the invention).

In a specific embodiment, the present invention relates to the targetedintegration of a promoter-less expression cassette into a chromosomaltranscription unit in T cells, preferably human T cells, to takeadvantage of an endogenous promoter to optimize transgene expression andenhance the function of the engineered T cells, wherein the transgene isa CAR or other therapeutic transgene. In a preferred embodiment, byengineering T cells this way, stable and homogenous CAR expression wasobtained, and T cell function and persistence was enhanced relative toprevious methods for CAR therapy. Depending on the cassette design, themethod can be used to disrupt, or not, the expression of the endogenousgene. In the case where endogenous gene expression is disrupted, theendogenous gene is a non-essential gene, i.e., a gene that is notnecessary for cell viability or proliferation of the cell. In aparticular embodiment, the therapeutic transgene is a CAR. In apreferred embodiment, the integration of the CAR-encoding nucleic acidsequences disrupts the expression of an endogenous gene encoding aprotein required for a functional T cell receptor complex. This approachcan be applied to any gene, having either stable, spatially, and/ortemporally regulated expression. In a specific embodiment, the targetingof a gene that is expressed from only one allele, for example, TCRalpha, TCR beta, Y or X chromosome-specific genes, can be utilized toensure that only one transgene copy per cell is expressed. Each T cellsexpresses a unique T cell receptor resulting from association of onerecombined TCR alpha and one recombined TCR beta chains. The process ofgenerating the TCR diversity happens during lymphopoiesis in the thymus,where both TCR alpha and beta genes recombine (VJ and VDJ recombinationrespectively), and only one allele of each gene is expressed through aprocess called allele exclusion (Honey, Nat. Rev. Immunol. 5, 95doi:10.1038/nri1560 (2005)). In the case of targeting a recombined TCRalpha or beta chain, this process provides that only one copy of theintegrated CAR will be expressed. The other allele can be targeted butwould not result in CAR expression.

The T cells of the invention are immune cells of the lymphoid lineage. Tcells express the T cell receptor (TCR), with most cells expressing αand β chains and a smaller population expressing γ and δ chains. T cellsuseful as immune cells of the invention can be CD4⁺ or CD8⁺ and caninclude, but are not limited to, T helper cells (CD4⁺), cytotoxic Tcells (also referred to as cytotoxic T lymphocytes, CTL; CD8⁻ T cells),and memory T cells, including central memory T cells (TCM), stem memoryT cells (TSCM), stem-cell-like memory T cells (or stem-like memory Tcells), and effector memory T cells, for example, T_(EM) cells andT_(EMRA) (CD45RA⁺) cells, effector T cells, Th1 cells, Th2 cells, Th9cells, Th17 cells, Th22 cells, Tfh (follicular helper) cells, Tregulatory cells, natural killer T cells, mucosal associated invariant Tcells (MAIT), and γδ T cells. Major T cell subtypes include T_(N)(naive), T_(SCM) (stem cell memory), T_(CM) (central memory), T_(TM)(Transitional Memory), T_(EM) (Effector memory), and T_(TE) (TerminalEffector). In one embodiment, the T cells of the invention areimmunostimulatory cells, i.e., cells that mediate an immune response.Exemplary T cells that are immunostimulatory include, but are notlimited to, T helper cells (CD4⁺), cytotoxic T cells (also referred toas cytotoxic T lymphocytes, CTL; CD8⁺ T cells), and memory T cells,including central memory T cells (TCM), stem memory T cells (TSCM),stem-cell-like memory T cells (or stem-like memory T cells), andeffector memory T cells, for example, T_(EM) cells and T_(EMRA)(CD45RA⁺) cells, effector T cells, Th1 cells, Th2 cells, Th9 cells, Th17cells, Th22 cells, Tfh (follicular helper) cells, natural killer Tcells, mucosal associated invariant T cells (MAIT), and γδ T cells. Inanother embodiment, the T cells of the invention are immunoinhibitorycells, i.e., cells that inhibit an immune response. Exemplary T cellsthat are immunoinhibitory include regulatory T cells (T regulatorycells, Treg) and follicular regulatory T cells (Tfh) cells. T cells canoptionally be generated from embryonic stem cells or induced pluripotentstem cells (iPSCs)(see, for example, Themeli et al., Nat. Biotechnol.31(10):928-933 (2013)). Optionally, precursor cells of T cells that canbe used, which recombinantly express a transgene, preferably a CAR, are,by way of example, hematopoietic stem and/or progenitor cells.Hematopoietic stem and/or progenitor cells can be derived from bonemarrow, umbilical cord blood, adult peripheral blood after cytokinemobilization, and the like, by methods known in the art, and then aregenetically engineered to recombinantly express a transgene, preferablya CAR. Particularly useful precursor cells are those that candifferentiate into the lymphoid lineage, for example, hematopoietic stemcells or progenitor cells of the lymphoid lineage that can differentiateinto T cells. In another embodiment, an iPSC can be utilized as a cellfor expression of a transgene. In a preferred embodiment, an iPSC can beutilized as a cell for expression of a CAR, wherein a recombinantnucleic acid encoding a CAR is integrated into a site in the genome ofthe cell such that the CAR is expressed by the cell at the surface ofthe cell, and wherein integration of the nucleic acid encoding the CARat the site reduces or prevents expression of a functional T cellreceptor complex at the surface of the cell. In another embodiment, a Tcell, preferably a CAR T cell, as disclosed herein can be used toproduce an iPSC. It is understood that embodiments disclosed hereinrelating to a T cell shall be deemed as applicable to an iPSC or stemcell, as context permits. An iPSC can be used to produce a T cell of theinvention, and an iPSC can also be derived therefrom.

The type of T cell selected for expressing a transgene will take intoconsideration whether it is desired to stimulate an immune response orinhibit an immune response. For example, a regulatory T cell (CD4+CD25high FoxpP3+) would be used for treating a subject in need of aninhibited immune response such as someone having an autoimmune diseaseand, by way of example, the T cell would express a transgene encoding animmuno-inhibitory cytokine, while CD4+ (except Treg)/CD8+ T cells areused to treat a subject in need of a stimulated immune response, forexample, a subject having cancer, and, by way of example, the T cellwould express an immunostimulatory cytokine.

T cells can be isolated by methods well known in the art, includingcommercially available isolation methods (see, for example,Rowland-Jones et al., Lymphocytes: A Practical Approach, OxfordUniversity Press, New York (1999)). Sources for the T cells include, butare not limited to, peripheral blood, umbilical cord blood, bone marrow,or other sources of hematopoietic cells. Various techniques can beemployed to separate the cells to isolate or enrich for desired immunecells such as T cells. For instance, negative selection methods can beused to remove cells that are not the desired immune cells.Additionally, positive selection methods can be used to isolate orenrich for desired T cells, or a combination of positive and negativeselection methods can be employed. Monoclonal antibodies (MAbs) areparticularly useful for identifying markers associated with particularcell lineages and/or stages of differentiation for both positive andnegative selections. If a particular type of T cell is to be isolated,various cell surface markers or combinations of markers, including butnot limited to, CD3, CD4, CD8, CD34 (for hematopoietic stem andprogenitor cells) and the like, can be used to separate the cells, as iswell known in the art (see Kearse, T Cell Protocols: Development andActivation, Humana Press, Totowa N.J. (2000); De Libero, T CellProtocols, Vol. 514 of Methods in Molecular Biology, Humana Press,Totowa N.J. (2009)).

Methods for isolating and expanding regulatory T cells are well known inthe art (see, for example, Su et al., Methods Mol. Biol. 806:287-299(2012); Bluestone et al., Sci. Transl. Med. 7(315) (doi:10.1126/scitranslmed.aad4134)(2015); Miyara et al., Nat. Rev. Rheumatol.10:543-551 (2014); Liu et al., J. Exp. Med. 203:1701-1711 (2006);Seddiki et al., J. Exp. Med. 203:1693-1700 (2006); Ukena et al., Exp.Hematol. 39:1152-1160 (2011); Chen et al., J. Immunol. 183:4094-4102(2009); Putnam et al., Diabetes 58:652-662 (2009); Putnam et al., Am.Tranplant. 13:3010-3020 (2013); Lee et al., Cancer Res. 71:2871-2881(2011); MacDonald et al., J Clin. Invest. 126:1413-1424 (2016)). Invitro generation of regulatory T cells (iTregs) has also been described(see, for example, Lan et al., J Mol. Cell. Biol. 4:22-28 (2012);Yamagiwa et al., J. Immunol. 166:7282-7289 (2001); Zheng et al., J.Immunol. 169:4183-4189 (2002)). Generally, regulatory T cells of theinvention are CD4⁺, for example, CD4⁺CD25⁺, and in particularCD4⁺CD127^(lo/−)CD25⁺. Such regulatory T cells express Foxp3 (forkheadbox P3), which is in the forkhead/winged-helix family of transcriptionfactors (Bluestone et al., J. Clin. Invest. 125:2250-2260 (2015); Rileyet al., Immunity 30:656-665 (2009)). A regulatory T cell that is animmunoinhibitory cell of the invention can also be a CD8⁺ regulatory Tcell (Guillonneau et al., Curr. Opin. Organ Transplant. 15:751-756(2010)). Methods for isolating and expanding regulatory T cells are alsocommercially available (see, for example, BD Biosciences, San Jose,Calif.; STEMCELL Technologies Inc., Vancouver, Canada; eBioscience, SanDiego, Calif.; Invitrogen, Carlsbad, Calif.). An immunoinhibitory T cellof the invention can also be a follicular regulatory T cell (T(FR))(Sage et al., Nat. Immunol. 14:152-161 (2013)). In a particularembodiment, the follicular regulatory T cells of the invention areCD4⁺CXCR5⁺ and express Foxp3 (Sage et al., supra, 2013).

Procedures for separation of cells include, but are not limited to,density gradient centrifugation, coupling to particles that modify celldensity, magnetic separation with antibody-coated magnetic beads,affinity chromatography; cytotoxic agents joined to or used inconjunction with a monoclonal antibody (mAb), including, but not limitedto, complement and cytotoxins, and panning with an antibody attached toa solid matrix, for example, a plate or chip, elutriation, flowcytometry, or any other convenient technique (see, for example,Recktenwald et al., Cell Separation Methods and Applications, MarcelDekker, Inc., New York (1998)).

The T cells can be autologous or non-autologous to the subject to whichthey are administered in the methods of treatment of the invention.Autologous cells are isolated from the subject to which the engineered Tcells are to be administered. In a preferred embodiment, autologouscells are isolated from the subject to which the engineered cellsrecombinantly expressing a CAR are to be administered. Optionally, thecells can be obtained by leukapheresis, where leukocytes are selectivelyremoved from withdrawn blood, made recombinant, and then retransfusedinto the donor. Alternatively, allogeneic cells from a non-autologousdonor that is not the subject can be used. In the case of anon-autologous donor, the cells are typed and matched for humanleukocyte antigen (HLA) to determine an appropriate level ofcompatibility, as is well known in the art. For both autologous and andnon-autologous cells, the cells can optionally be cryopreserved untilready to be used for genetic manipulation and/or administration to asubject using methods well known in the art.

Various methods for isolating T cells that can be used for recombinantexpression of a CAR have been described previously, and can be used,including but not limited to, using peripheral donor lymphocytes(Sadelain et al., Nat. Rev. Cancer 3:35-45 (2003); Morgan et al.,Science 314:126-129 (2006), using lymphocyte cultures derived from tumorinfiltrating lymphocytes (TILs) in tumor biopsies (Panelli et al., JImmunol. 164:495-504 (2000); Panelli et al., J Immunol. 164:4382-4392(2000)), and using selectively in vitro-expanded antigen-specificperipheral blood leukocytes employing artificial antigen-presentingcells (AAPCs) or dendritic cells (Dupont et al., Cancer Res.65:5417-5427 (2005); Papanicolaou et al., Blood 102:2498-2505 (2003)).In the case of using stem cells, the cells can be isolated by methodswell known in the art (see, for example, Klug et al., Hematopoietic StemCell Protocols, Humana Press, New Jersey (2002); Freshney et al.,Culture of Human Stem Cells, John Wiley & Sons (2007)).

In a specific embodiment, isolated T cells are genetically engineered exvivo for recombinant expression of a transgene. In a preferredembodiment, isolated T cells are genetically engineered ex vivo forrecombinant expression of a CAR. The cells can be genetically engineeredfor recombinant expression by methods well known in the art.

In another embodiment, the invention provides T cells that recognize andare sensitized to a target antigen that are then genetically engineeredfor recombinant expression of a transgene. Such T cells can but need notexpress a CAR that binds to a target antigen, since the cells alreadyare target antigen-specific so that their immune response (for example,cytotoxicity) is stimulated specifically by such target antigen. Such Tcells that recognize and are sensitized to a target antigen can beobtained by known methods, by way of example, in vitro sensitizationmethods using naive T cells (see, for example, Wolfl et al., Nat.Protocols 9:950-966 (2014)) or hematopoietic progenitor cells (see vanLent et al., J Immunol. 179:4959-4968 (2007)); or obtained from asubject that has been exposed to and is mounting an immune responseagainst the target antigen (i.e., in vivo sensitized T cells). Methodsfor isolating an antigen-specific T cell from a subject are well knownin the art. Such methods include, but are not limited to, a cytokinecapture system or cytokine secretion assay, which is based on thesecretion of cytokines from antigen stimulated T cells that can be usedto identify and isolate antigen-specific cells, and expansion of cellsin vitro (see Assenmacher et al., Cytometric Cytokine Secretion Assay,in Analyzing T Cell Responses: How to Analyze Cellular Immune ResponsesAgainst Tumor Associated Antigens, Nagorsen et al., eds., Chapter 10,pp. 183-195, Springer, The Netherlands (2005); Haney et al., J. Immunol.Methods 369:33-41 (2011); Bunos et al., Vox Sanguinis DOI:10.1111/vox.12291 (2015); Montes et al., Clin. Exp. Immunol. 142:292-302(2005); Adusumilli et al., Sci Transl Med. 6:261ra151 (2014)). Suchcytokines include, but are not limited to interferon-γ and tumornecrosis factor-α. Methods for isolating an antigen-specific regulatoryT cell from a subject are well known in the art (see, for example, Noyanet al., Eur. J. Immunol. 44:2592-2602 (2014); Brusko et al., PLoS One5(7) e11726 (doi: 10.1371) (2010); Bacher et al., Mucosal Immunol.7:916-928 (2014); Koenen et al., J. Immunol. 174:7573-7583 (2005)). Theantigen-specific T cells can be isolated using well known techniques asdescribed above for isolating T cells, which include, but are notlimited to, flow cytometry, magnetic beads, panning on a solid phase,and so forth. Antigen-specific T cell isolation techniques are alsocommercially available, which can be used or adapted for clinicalapplications (see, for example, Miltenyi Biotec, Cambridge, Mass.;Proimmune, Oxford, UK; and the like).

The T cells can be subjected to conditions that favor maintenance orexpansion of the cells (see Kearse, T Cell Protocols: Development andActivation, Humana Press, Totowa N.J. (2000); De Libero, T CellProtocols, Vol. 514 of Methods in Molecular Biology, Humana Press,Totowa N.J. (2009); Parente-Pereira et al., J. Biol. Methods 1(2) e7(doi 10.14440/jbm.2014.30) (2014); Movassagh et al., Hum. Gene Ther.11:1189-1200 (2000); Rettig et al., Mol. Ther. 8:29-41 (2003); Agarwalet al., J. Virol. 72:3720-3728 (1998); Pollok et al., Hum. Gene Ther.10:2221-2236 (1999); Quinn et al., Hum. Gene Ther. 9:1457-1467 (1998);Su et al., Methods Mol. Biol. 806:287-299 (2012); Bluestone et al., Sci.Transl. Med. 7(315) (doi: 10.1126/scitranslmed.aad4134)(2015); Miyara etal., Nat. Rev. Rheumatol. 10:543-551 (2014); Liu et al., J. Exp. Med.203:1701-1711 (2006); Seddiki et al., J. Exp. Med. 203:1693-1700 (2006);Ukena et al., Exp. Hematol. 39:1152-1160 (2011); Chen et al., J.Immunol. 183:4094-4102 (2009); Putnam et al., Diabetes 58:652-662(2009); Putnam et al., Am. J. Tranplant. 13:3010-3020 (2013); Lee etal., Cancer Res. 71:2871-2881 (2011); MacDonald et al., J. Clin. Invest.126:1413-1424 (2016); see also commercially available methods such asDynabeads™ human T cell activator products, Thermo Fisher Scientific,Waltham, Mass.)). The cells can optionally be expanded prior to or afterex vivo genetic engineering. Expansion of the cells is particularlyuseful to increase the number of cells for administration to a subject.Such methods for expansion of immune cells such as T cells are wellknown in the art (see Kaiser et al., Cancer Gene Therapy 22:72-78(2015); Wolfl et al., Nat. Protocols 9:950-966 (2014)). Furthermore, thecells can optionally be cryopreserved after isolation and/or geneticengineering, and/or expansion of genetically engineered cells (seeKaiser et al., supra, 2015)). Methods for cyropreserving cells are wellknown in the art (see, for example, Freshney, Culture of Animal Cells: AManual of Basic Techniques, 4th ed., Wiley-Liss, New York (2000);Harrison and Rae, General Techniques of Cell Culture, CambridgeUniversity Press (1997)).

7.2 Targeted Integration Methods

With respect to generating cells recombinantly expressing a transgeneunder control of an endogenous T cell promoter, the transgene isintroduced into the genome of the T cell. In a preferred embodiment,with respect to generating cells recombinantly expressing a CAR, anucleic acid encoding the CAR is introduced into the T cell.Traditionally, such methods have utilized a suitable expression vector,in which case the T cells are transduced with a transgene, for example,a nucleic acid encoding a CAR. In the present invention, a transgene iscloned into a targeting construct, which provides for targetedintegration of the transgene at a site within the genome. In a preferredembodiment, a nucleic acid encoding a CAR is cloned into a targetingconstruct, which provides for targeted integration of the nucleic acidsequence encoding the CAR at a site within the genome, in a particularembodiment, a site that disrupts expression of a gene encoding a proteinrequired for expression of a functional TCR complex in the cell. Forexample, a transgene, for example, a polynucleotide encoding a CAR, ofthe invention can be cloned into a suitable targeting construct, or asuitable vector such as a retroviral vector, and introduced into the Tcell using well known molecular biology techniques (see Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1999)).

Any suitable targeting construct suitable for expression in a cell ofthe invention, particularly a human T cell, can be employed. In aparticular embodiment, the targeting construct is compatible for usewith a homologous recombination system suitable for targeted integrationof the nucleic acid sequence (transgene) at a site within the genome ofthe cell. Exemplary homologous recombination systems are well known inthe art and include, but are not limited to, technologies utilizing anuclease, for example, transcription activator-like effector nucleases(TALENs), Zinc-finger nucleases (ZFNs), clustered regularly interspacedshort palindromic repeats (CRISPRs) systems such as and CRISPRassociated protein 9 (Cas9) and Cpf1, and/or Meganuclease or a Mega-Tal(fusion of a Tal domain and a Meganuclease) and the like, which providefor homologous recombination, for example, a desired target site withinthe genome of the cell (see Examples; see also U.S. Pat. No. 8,697,359;US publication 20140068797; Gaj et al., Trends Biotechnol. 31:397-405(2013); Gersbach et al., Nucl. Acids Res. 39:7868-7878 (2011); Vasileva,et al. Cell Death Dis. 6:e1831. (Jul. 23 2015); Sontheimer, Hum. GeneTher. 26(7):413-424 (2015); Osborn et al., Mol. Ther. 24(3):570-581(2016))). Such methods are well known in the art and commerciallyavailable (ThermoFisher, Carlsbad, Calif.; GenScript, Piscataway, N.J.;Clontech, Mountain View, Calif.). Other CRISPR based systems includepyrogen and Aureus. Such methods can be used to carry out or promotehomologous recombination.

7.3 Vectors and Targeting Constructs

A suitable targeting construct can comprise any nucleic acid sequencethat is compatible with a homologous recombination system employed inthe invention. In one embodiment, the targeting construct comprisesadeno-associated virus (AAV) sequences. The targeting construct can havenucleic acid sequences from one or more AAV serotypes. For example, thetargeting construct can comprise AAV2 sequences or other serotypesequences such as AAVS. The AAV nucleic acid sequences that function aspart of a targeting construct can be packaged in several natural orrecombinant AAV capsids or particles. In a particular embodiment, theAAV particle is AAV6. In a particular embodiment, an AAV2-basedtargeting construct is delivered to the target cell using AAV6 viralparticles. In a particular embodiment, the AAV sequences are AAV2, AAVSor AAV6 sequences. In a particular embodiment, the AAV sequences arefrom AAV2. In another particular embodiment, the AAV sequences are fromAAV6. In another particular embodiment, the targeting constructcomprises in 5′ to 3′ order: a first viral sequence, a left homologyarm, a nucleic acid sequence encoding a self-cleaving porcineteschovirus 2A, a transgene, a polyadenylation sequence, a righthomology arm and a second viral sequence. In a preferred embodiment, thetargeting construct comprises in 5′ to 3′ order: a first viral sequence,a left homology arm, a nucleic acid sequence encoding a self-cleavingporcine teschovirus 2A, a nucleic acid sequence encoding a CAR, apolyadenylation sequence, a right homology arm and a second viralsequence. Another suitable targeting construct can comprise sequencesfrom an integrative-deficient Lentivirus (see, for example, Wanisch etal., Mol. Ther. 17(8):1316-1332 (2009)). In a particular embodiment, theviral nucleic acid sequence comprises sequences of anintegrative-deficient Lentivirus. It is understood that any suitabletargeting construction compatible with a homologous recombination systememployed can be utilized.

Viral vector sequences that can be included in a target constructinclude but are not limited to retroviral, adenoviral, lentiviral, andadeno-associated viral vectors, vaccinia virus, bovine papilloma virusderived vectors, and herpes virus vectors, such as Epstein-Barr Virus(see, for example, Miller, Hum. Gene Ther. 1(1):5-14 (1990); Friedman,Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614(1988); Tolstoshev et al., Current Opin. Biotechnol. 1:55-61 (1990);Sharp, Lancet 337:1277-1278 (1991); Cornetta et al., Prog. Nucleic AcidRes. Mol. Biol. 36:311-322 (1989); Anderson, Science 226:401-409 (1984);Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993);and Johnson, Chest 107:77S-83S (1995); Rosenberg et al., N. Engl. J.Med. 323:370 (1990); Anderson et al., U.S. Pat. No. 5,399,346; Scholleret al., Sci. Transl. Med. 4:132-153 (2012; Parente-Pereira et al., J.Biol. Methods 1(2):e7 (1-9)(2014); Lamers et al., Blood 117(1):72-82(2011); Reviere et al., Proc. Natl. Acad. Sci. USA 92:6733-6737 (1995);Wang et al., Gene Therapy 15:1454-1459 (2008)).

Particularly useful vectors for generating a target construct thatprovides transgene vectorization for homologous recombination-mediatedtargeting include, but are not limited to, recombinant Adeno-AssociatedVirus (rAAV), recombinant non-integrating lentivirus (rNILV),recombinant non-integrating gamma-retrovirus (rNIgRV), single-strandedDNA (linear or circular), and the like. Such vectors can be used tointroduce a transgene into a T cell of the invention by making a targetconstruct, as described above.

In one embodiment, the vector is a recombinant non-integratinggamma-retrovirus (rNIgRV). In one embodiment, the rNIgRVs is obtained byusing a gamma-retrovirus integrase that is mutated at the DDE motif,which abolishes integrase activity. Thus, a gamma-retrovirus isconverted to a non-integrating gamma-retrovirus by inactivation of itsintegrase (see Example 4 and FIG. 18). In a particular embodiment, theintegrase comprises a DDE mutation selected from the group consisting ofD164A, D164E, D164N, D164V, D183A, D183N, D164A and D168A, D164A andD183N, D164N and D183A, D164N and D183N, D164V and D168A, D164V andD183N, D164V and D183A, and D164V and D183N. Such a rNIgRV vector isadvantageous since it is easier and cheaper to produce thantraditionally used vectors.

It will be readily understood that a rNIgRV vector can be utilized tointroduce any desired DNA into any cell. Thus, a rNIgRV can be used tointroduce any type of desired DNA into a cell of any type in which thevector functions.

In methods of the present invention that employ an endogenous promoterfor controlling the expression of a transgene that is integrated withina site in the genome of a cell, the targeting construct preferably ispromoter-less. In a preferred embodiment of methods of the presentinvention that employ an endogenous promoter for controlling theexpression of a nucleic acid sequence encoding a CAR that is integratedwithin a site in the genome of a cell, the targeting constructpreferably is promoter-less. Such a construct allows the integration ofthe transgene, such as the nucleic acid sequence encoding a CAR, into asite within the genome such that the integrated nucleic acid sequence(transgene) is under the control of an endogenous promoter. In oneembodiment, the endogenous promoter is a TCR promoter. In a particularembodiment, the endogenous promoter is a promoter of a gene encoding a Tcell receptor alpha chain, T cell receptor beta chain, CD3 gamma chain,CD3 delta chain, CD3 epsilon chain, or CD3 zeta chain. In a specificembodiment, the nucleic acid sequences encoding a CAR are integrated.

Although the methods of the invention preferably utilize an endogenouspromoter to control expression of the recombinant transgene, such as thenucleic acid sequence encoding a CAR, it is understood that a vectorthat employs a suitable promoter for expression in a particular hostcell can be utilized, for example, a vector that incorporates anendogenous promoter such as a TCR promoter. Such a vector could providefor expression in a manner similar to that provided by an endogenouspromoter, such as a TCR promoter. Such a vector can be useful, forexample, if the site of integration does not provide for efficientexpression of a transgene, or if disruption of the endogenous genecontrolled by the endogenous promoter would be detrimental to the T cellor would result in a decrease in its effectiveness in T cell therapy. Ina preferred embodiment, such a vector can be useful, for example, if thesite of integration does not provide for efficient expression of nucleicacid sequence encoding a CAR. The promoter can be an inducible promoteror a constitutive promoter. Expression of a nucleic acid sequence underthe control of an endogenous or vector-associated promoter occurs undersuitable conditions for the cell to express the nucleic acid, forexample, growth conditions, or in the presence of an inducer with aninducible promoter, and the like. Such conditions are well understood bythose skilled in the art.

The targeting construct can optionally be designed to include a P2Asequence directly upstream of the nucleic acid sequences encoding thetransgene. In a preferred embodiment, the targeting construct canoptionally be designed to include a P2A sequence directly upstream ofthe nucleic acid sequences encoding a CAR. P2A is a self-cleavingpeptide sequence, which can be used for bicistronic or multicistronicexpression of protein sequences (see Szymczak et al., Expert Opin. Biol.Therapy 5(5):627-638 (2005)). If desired, the targeting construct canoptionally be designed to include a reporter, for example, a reporterprotein that provides for identification of transduced cells. Exemplaryreporter proteins include, but are not limited to, fluorescent proteins,such as mCherry, green fluorescent protein (GFP), blue fluorescentprotein, for example, EBFP, EBFP2, Azurite, and mKalamal, cyanfluorescent protein, for example, ECFP, Cerulean, and CyPet, and yellowfluorescent protein, for example, YFP, Citrine, Venus, and YPet.

Preferably, the targeting construct comprises a polyadenylation (poly A)sequence 3′ of the transgene. In a preferred embodiment, the targetingconstruct comprises a polyadenylation (poly A) sequence 3′ of thenucleic acid sequences encoding a CAR.

As disclosed herein, in a specific embodiment, a nucleic acid encoding aCAR is integrated at a site within the genome of the cell such that theCAR can be expressed in the cell and produced at the cell surface. Thesite of integration reduces or prevents expression of a functional Tcell receptor (TCR) complex at the surface of the cell. The cell therebycan become a TCR negative cell. Such a TCR negative cell can be useful,for example, in the case of utilizing non-autologous T cells, forreducing graft versus host disease (GVHD) in the recipient. Generating aTCR negative cell also can be used to treat a subject having anautoimmune disease with autologous cells, since the autoimmune reactionprovided by the subject's own T cells can be reduced by reducing orpreventing expression of a functional TCR complex that targets anautoantigen.

The T cell receptor (TCR) is a heterodimer of TCR-α and TCR-β chains.The TCR complex is formed by TCR and CD3 gamma (γ), CD3 delta (δ), CD3epsilon (ε), and CD3 zeta (ζ) (see, for example, Call et al., Cell111:967-979 (2002)). Disruption or reduced expression of one or more ofa T cell receptor alpha chain, T cell receptor beta chain, CD3 gammachain, CD3 delta chain, CD3 epsilon chain, or CD3 zeta chain can be usedto reduce or prevent formation of a functional T cell receptor (TCR)complex. By reducing or preventing the formation of a functional TCRcomplex, the T cell no longer mediates an immune response through itsTCR complex. In one embodiment, a nucleic acid encoding a CAR isintegrated at a site within the genome that disrupts or reduces theexpression of a T cell receptor alpha chain, T cell receptor beta chain,CD3 gamma chain, CD3 delta chain, CD3 epsilon chain, or CD3 zeta chain.While the reduction of one of the TCR complex proteins can besufficient, it is understood that more than one component of the TCRcomplex can be reduced, if desired.

It is understood that the site of integration in the genome of the cellis targeted to place the transgene under control of an endogenouspromoter. The integration can be, by way of example but not limitation,integration into an exon, integration into an intron, or integration atthe 5′ end of the gene. In one embodiment, integration of the transgeneresults in disruption of the endogenous gene at the site of integration.In a preferred embodiment, it is understood that the site of integrationin the genome of the cell is targeted to reduce or disrupt expression ofa component of the TCR complex, for example, T cell receptor alphachain, T cell receptor beta chain, CD3 gamma chain, CD3 delta chain, CD3epsilon chain, or CD3 zeta chain. One skilled in the art can readilydetermine a suitable position within a gene encoding a T cell receptoralpha chain, T cell receptor beta chain, CD3 gamma chain, CD3 deltachain, CD3 epsilon chain, or CD3 zeta chain to integrate a CAR encodingnucleic acid to reduce or disrupt expression of T cell receptor alphachain, T cell receptor beta chain, CD3 gamma chain, CD3 delta chain, CD3epsilon chain, or CD3 zeta chain. Such methods are well known in the artand can include, but are not limited to, integration into an exon,integration into an intron, integration at the 5′ end of the gene, andthe like. It is understood that any intron or exon of the gene cansupport the targeting construct. One skilled in the art can readilydetermine a suitable site for targeted integration of a transgene that,if desired, will reduce or disrupt expression of an endogenous geneunder control of the endogenous promoter at the site of integration. Ina particular embodiment, the site of integration is within the firstexon. It is understood that, when selecting a site for integration of atransgene, the integration site occurs in a non-essential gene, i.e., agene that is not necessary for cell viability or proliferation of thecell, particularly in the case where expression of the endogenous genewill be disrupted. In a preferred embodiment, one skilled in the art canreadily determine a suitable site for targeted integration of a nucleicacid sequence encoding a CAR that will reduce or disrupt expression ofTCR complex protein such as T cell receptor alpha chain, T cell receptorbeta chain, CD3 gamma chain, CD3 delta chain, CD3 epsilon chain, or CD3zeta chain, and/or place the CAR encoding nucleic acid sequence underthe control of the endogenous promoter of the respective gene encodingthe TCR complex component. In one embodiment, the site of integration iswithin the first exon (see Example). In a particular embodiment, thesite of integration is within the first exon of the TCR alpha constantchain (TRAC). In a preferred embodiment, a transgene, such as a nucleicacid encoding a CAR, is placed under control of an endogenous TCRpromoter. Details thereof are described in provisional application Nos.62/323,623, filed Apr. 15, 2016, and 62/323,675, filed Apr. 16, 2106,which are incorporated herein by reference in their entireties.

If desired, the integration site and targeting construct can be designedto provide integration of a transgene in frame with the endogenous gene,resulting in expression of a fusion protein of the transgene and theendogenous gene (see also US20130280222). In a preferred embodiment, theintegration site and targeting construct can be designed to provideintegration in frame with the endogenous gene, resulting in expressionof a fusion protein of a CAR and the TCR complex protein. Optionally,such a construct can contain a P2A directly 5′ of the transgene,allowing the expression of the transgene at a desired location in thecell without being fused to the gene product of the endogenous gene.Such a construct provides for expression of both the transgene and theendogenous gene at the site of integration, and such a construct can beutilized if disruption of the endogenous gene is detrimental to the Tcell or would result in a decrease in its effectiveness in T celltherapy. In a preferred embodiment, such a construct can contain a P2Adirectly 5′ of the nucleic acid sequence encoding the CAR, allowing theexpression of the CAR at the surface of the cell without being fused toTCR complex protein. It is further understood that another gene also canbe integrated into the genome such as a gene encoding a second CAR, or asafety switch (e.g., inducible caspase 9 (iCasp9) or herpes simplexvirus thymidine kinase (HSVtk), see Tey, Clin. Transl. Immunology3(6):e17), or an immunomodulatory molecule, and the like. In oneembodiment, integration of the same or different genes (transgenes)occurs in different target genes, respectively. In a specific aspect,different genes (transgenes) are integrated at the different integrationsites, respectively.

The homologous recombination system is designed using methods well knownin the art to target a desired site within the genome, for example, asite within the gene encoding T cell receptor alpha chain (Chromosome14, NC_000014.9 (22547506 . . . 22552132)), T cell receptor beta chain(Chromosome 7, NC_000007.14 (142299011 . . . 142813287)), CD3 gammachain (Chromosome 11, NC_000011.10 (118344316 . . . 118353782)), CD3delta chain (Chromosome 11, NC_000011.10 (118339074 . . . 118342744)),CD3 epsilon chain (Chromosome 11, NC_000011.10 (118304580 . . .118316175)), or CD3 zeta chain (Chromosome 1, NC_000001.11 (167430640 .. . 167518616)), as is known in the art (Chromosome location numberscorrespond to the current assembly: GRCh38.p2).

As described herein, in one embodiment, the integration site can targeta gene that is expressed from only one allele, for example, TCR alpha,TCR beta, Y or X chromosome-specific genes. In such a case, it can besufficient to integrate a transgene at a single site within the genome.In a preferred embodiment wherein the transgene encodes a CAR, in such acase, it can be sufficient to integrate a nucleic acid encoding a CAR ata single site within the genome. This strategy can be utilized to ensurethat only one transgene copy per cell is expressed. Optionally, in thecase where a gene to be targeted for integration is present on twoalleles, targeted homologous recombination can occur at both alleles. Insuch a case, the targeted integration can occur at one locus or twoloci.

Assays can be used to determine the transduction efficiency of atransgene, preferably encoding a CAR, using routine molecular biologytechniques. Gene transfer efficiency can be monitored by fluorescenceactivated cell sorting (FACS) analysis to quantify the fraction oftransduced T cells, and/or by quantitative PCR. Using a well-establishedcocultivation system (Gade et al., Cancer Res. 65:9080-9088 (2005); Gonget al., Neoplasia 1:123-127 (1999); Latouche et al., Nat. Biotechnol.18:405-409 (2000)) it can be determined whether fibroblast AAPCsexpressing cancer antigen (vs. controls) direct cytokine release fromtransduced T cells expressing a CAR (cell supernatant LUMINEX (AustinTex.) assay for IL-2, IL-4, IL-10, IFN-γ, TNF-α, and GM-CSF), T cellproliferation (by carboxyfluorescein succinimidyl ester (CFSE)labeling), and T cell survival (by Annexin V staining). T cellsexpressing a CAR can be exposed to repeated stimulation by targetantigen positive cells, and it can be determined whether T cellproliferation and cytokine response remain similar or diminished withrepeated stimulation. In a preferred embodiment, T cells expressing aCAR can be exposed to repeated stimulation by cancer antigen positivetarget cells, and it can be determined whether T cell proliferation andcytokine response remain similar or diminished with repeatedstimulation. Cytotoxicity assays with multiple E:T ratios can beconducted using chromium-release assays.

7.4 Endogenous T Cell Promoters

The invention relates to expressing a therapeutic transgene in a T cellby integrating the transgene at a site within the genome of the T cellsuch that the transgene is placed under the control of an endogenouspromoter of the T cell. By utilizing an endogenous promoter, T cells areengineered to express a therapeutic transgene, or a variety oftherapeutic transgenes under the control of different endogenouspromoters. In a specific embodiment, expression of the transgene isdependent on the microenvironment of the T cell. For example, expressionof a therapeutic transgene can be made dependent on the location of theT cell (e.g., expression of a transgene only in proximity to a tumor) byusing an endogenous promoter that is induced when the T cell is at aparticular location (e.g., when the T cell is at the location of a tumorand is activated by binding to tumor antigen, thereby inducing theendogenous promoter), or can be at defined time points (e.g., by usingan endogenous promoter that is induced at a defined time point, e.g. byactivation of the T cell upon encountering a tumor cell). The promoteris selected based on, for example, how soon it is activated or inhibitedafter encounter of the T cell with an antigen, how strongly it isexpressed, and for how long. The promoter is selected to accommodate thepharmacology for the transgene whose expression it regulates (e.g., sometransgenes are more effective at low levels, other transgenes are moreeffective at high levels of expression, and the like). It will beunderstood that the description in this disclosure with respect to useof an endogenous promoter (singular) controlling the expression of atransgene in a T cell will apply equally to the use of more than oneendogenous promoter, each controlling the expression of a transgene(that can be the same or different from the other transgenes), in the Tcell, unless context indicates otherwise. One skilled in the art canreadily select appropriate endogenous promoters to provide desiredexpression and/or regulation of one or more transgenes to enhance theeffectiveness of a T cell for use in T cell therapy.

The endogenous T cell promoters can be constitutive or inducible. In aspecific embodiment, the endogenous promoter is specific for a subset ofT cells. In the case where more than one transgene is expressed in a Tcell, the transgenes (which may be different from each other) can beplaced under control of a combination of constitutive and induciblepromoters, respectively, of which one or more can be, for example,specific for a subset of T cells.

In one aspect of the embodiments described herein, the endogenouspromoter is not an interleukin 4 (IL4) promoter.

In one embodiment, the endogenous T cell promoter is constitutive. Inanother embodiment, the endogenous T cell promoter is inducible. In aspecific embodiment, the endogenous T cell promoter is active in asubset of T cells. In one embodiment, two or more transgenes areintegrated into the genome of the T cell, such that expression of eachtransgene is under the control of a different endogenous promoter of theT cell. In a specific embodiment, two transgenes are thus integrated. Ina particular embodiment, the expression of each of two transgenes isunder the control of different endogenous promoters that areconstitutive. In another particular embodiment, the expression of eachof two transgenes is under the control of different endogenous promotersthat are inducible. In another particular embodiment, the expression ofa first transgene is under control of a constitutive endogenous promoterand expression of a second transgene is under control of an inducibleendogenous promoter. In another particular embodiment, three transgenesare integrated into the genome of the T cell, such that expression ofeach transgene is under the control of a different endogenous promoterof the T cell, where expression of a first transgene is under control ofa constitutive endogenous promoter and expression of second and thirdtransgenes is under control of two different inducible, endogenouspromoters, respectively. It is understood that, depending on thetransgene to be expressed in the T cell, a promoter can be selected toprovide an appropriate expression level, time of expression, expressionwhen the T cell is in a particular microenvironment, and so forth. Forexample, expression of transgene 1 can be under control of aconstitutive promoter, expression of transgene 2 can be under control ofan inducible promoter that is activated shortly after contact with anantigen recognized by the T cell, and expression of transgene 3 can beunder control of a different inducible promoter that is activated at alater time or at a different level than for transgene 2. In thisparticular example, transgene 1 is expressed constitutively, andtransgenes 2 and 3 are under control of inducible promoters withdistinct characteristics.

Engineering of T cells of the invention to express a transgene from anendogenous T cell promoter provides for autonomous regulation oftransgene expression by the T cell. Thus, the microenvironment of the Tcell can be used to coordinate the expression of multiple transgenes toprovide optimized activity of the transgenic T cell, particularly whenat least one gene is under control of an inducible promoter. Forexample, T cell therapy can be accompanied by administration of a T cellstimulatory cytokine (see Sadelain et al., Cancer Disc. 3:388-398(2013)). In one embodiment, the T cells of the invention can beengineered to co-express a CAR and a second transgene, such as a T cellactivating cytokine. For example, a CAR can be placed under control of aconstitutive promoter, and a second transgene such as a T cellactivating cytokine (e.g., interleukin 12 (IL12)) can be placed undercontrol of an inducible promoter such that activation of the induciblepromoter controlling the second transgene occurs when the T cell is inproximity to an antigen recognized by the CAR such as on a tumor, forexample, when the T cell engages a target tumor antigen by binding tothe CAR. In this example, such a construct obviates the need forsystemic or localized administration of a T cell activating cytokine,which can result in toxicity. In addition, in the case where the T cellis engineered to express a T cell activation cytokine under control ofan inducible promoter that can be regulated by administration of a drug,such a construct obviates the need to administer the drug. In such acase, instead of needing to administer a drug to induce expression of atransgene, regulation of transgene expression is under control of anendogenous promoter, which provides for expression of the transgene.Instead, the T cell itself, upon engagement with a target antigen,activates expression of a T cell activating cytokine, providinglocalized expression of the cytokine, and therefore spatio-temporalregulation of expression of transgenes to optimize the effectiveness ofthe T cells to be used for immunotherapy.

In another example, a T cell expressing a CAR can sometimes exhibittoxicities. To reduce such toxicity, in a specific embodiment, atransgene encoding a CAR can therefore be placed under control of aninducible promoter such that the promoter is not induced, and expressionof the CAR does not occur, until the T cell is engaged with a targetrecognized by the CAR, such as a target tumor. In yet anotherembodiment, a T cell can be engineered to have higher selectivity for aparticular target. For example, in some cases a target antigen on atumor may not be expressed on the tumor only. Therefore, targeting of aT cell to the target antigen could result in an immune response againstnon-target cells or tissues that express the same antigen. Accordingly,in one embodiment, a T cell of the invention is engineered to recognizetwo antigens on a target tumor, which provides higher selectivity forthe target tumor. For example, the T cell can be engineered to expresstwo CARs specific for two different tumor antigens. In this case,selective binding of the T cell to a target bearing two target antigenscan be coupled with a third transgene under control of an inducibleendogenous promoter, such as a T cell activating cytokine as describedabove, thereby stimulating activation of the T cell with the cytokineonly upon selective engagement with the target. A person skilled in theart will readily understand that selection of suitable therapeutictransgenes to be expressed under the control of suitable endogenous Tcell promoters, either constitutive, specific for a subtype of T cells,inducible, or a combination thereof, can be used to generateautonomously regulated expression of transgenes to provide moreeffective T cell therapy. In one embodiment, instead of using a fullycompetent CAR targeting one antigen, two sub-optimal CAR targeting twodifferent antigens need to be engaged for a full antitumor response. Ifhealthy tissues express one or the other antigen, they healthy tissuewill not fully engage a CAR T cell response. If the tumor expresses thetwo antigen, it will then trigger a complete CAR T cell activity.

The invention relates to optionally using both constitutive andinducible promoters, since a T cell can be engineered to specificallyrespond to a particular molecular cue to produce new therapeuticmolecules at a chosen location and time. For example, a transgeneencoding an antigen-specific cell-surface receptor can be expressed froma constitutive promoter and will only signal upon interaction with thatparticular antigen. Then, this interaction induces the activation of aspecific promoter that controls the expression of a therapeuticmolecule. The therapeutic benefit of this particular engineered T celldepends on the function of both constitutive and inducible promoters. Ina particular embodiment, a CAR can be under the under the control of aconstitutive promoter (e.g., TRAC, CD3s, B2M . . . ). In a particularembodiment, another therapeutic transgene (monoclonal antibody(checkpoint inhibitor, and the like) or cytokines (e.g., IL12, IL18 andthe like) are under the control of promoter activated by CAR engagement(e.g., IL2, IFNg, CD69 . . . ). In such a case, the transgene would beexpressed upon CAR activation and specifically be expressed in thetumor.

In one embodiment, the invention relates to expressing 3 transgenes, ormore. For example, transgene 1 can be constitutive, transgene 2 can comein shortly after contact with antigen, and transgene 3 can come on lateror at a different level than transgene 2. In this example, expression oftransgene 1 is under the control of an endogenous constitutive promoter,expression of transgene 2 begins shortly after contact with antigen byvirtue of being controlled by an endogenous promoter induced by antigenengagement, and expression of transgene 3 begins later or at a differentlevel than transgene 2 by virtue of being controlled by an endogenouspromoter induced later or providing for a different level of expressionthan the endogenous promoter regulating transgene 2. In this example,transgene 1 is constitutive and transgenes 2 and 3 are inducible (eachwith distinct kinetic characteristics). In a particular embodiment,transgene 1 encodes a CAR specific for antigen A, e.g., on tumor cells,where transgene 1 is constitutively expressed. After binding to antigenA, transgene 2 is expressed, which encodes another CAR specific forantigen B (e.g., also expressed on tumor cells or on other cells withinthe tumor microenvironment). Transgene 3 can be, for example, a thirdCAR; this third CAR can recognize antigen C, e.g., also on the tumorcells or other cells within the tumor microenvironment. This example isa form of “combinatorial targeting” using temporal/sequential expressionof different CARs by the same T cell. In another particular embodiment,transgene 1 encodes a CAR (or TCR) specific for antigen A; transgene Bencodes a cytokine, and transgene 3 encodes another cytokine or acostimulatory ligand or an scFv, for example, recognizing an antigen onthe same cells (e.g., tumor cells) that express antigen A or cells inthe same microenvironment. This is an example of sequential geneactivation designed to increase T cell potency and safety by confininggene expression to a microenvironment such as the tumormicroenvironment. Thus, a person skilled in the art can select anendogenous T cell promoter for placement of a desired transgene toprovide desired expression characteristics of the transgene.

It is further understood that certain transgenes (e.g.,immunostimulatory transgenes—those that when expressed provide animmunostimulatory effect) are desirable to express in a T cell that isimmunostimulatory, whereas other transgenes (e.g., immunoinhibitorytransgenes—those that when expressed provide an immunoinhibitory effect)are desirable to express in a T cell that is immunoinhibitory. It isunderstood that a person skilled in the can readily determine suitabletransgenes to express in a T cell depending on whether it is desired tostimulate or inhibit an immune response. As will be clear, in preferredembodiments, an immunostimulatory transgene is expressed in animmunostimulatory T cell to stimulate an immune response in the subjectto which the T cell is administered, and an immunoinhibitory transgeneis expressed in an immunoinhibitory T cell to inhibit an immune responsein the subject to which the T cell is administered.

Constitutive Promoters. In one embodiment, a therapeutic transgene isintegrated at a site within the genome of a T cell such that expressionof the transgene is placed under control of an endogenous promoter thatis constitutive. The constitutive promoters can be used to express atransgene such as a CAR or CCR to activate the immune response. Aconstitutive promoter can also be used to inhibit an immune response ifcontrols expression of an inhibitory CAR (iCAR) containing PD1 and orcTLA4 intracellular domain, and the like.

In one embodiment, a constitutive promoter is a TCR promoter, i.e., apromoter of a protein of the T cell receptor complex (TCR) (seeExamples). In a particular embodiment, the endogenous promoter is apromoter of a gene encoding a T cell receptor alpha chain, T cellreceptor beta chain, CD3 gamma chain, CD3 delta chain, CD3 epsilonchain, or CD3 zeta chain.

In another embodiment, a constitutive promoter can be, but is notlimited to, a promoter such as CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter, or the like (see also Table 1,promoters indicated as constitutive).

TABLE 1 Exemplary Constitutive and Inducible Promoters and CorrespondingInducers. T cells subset Promoter Inducer Immunue response Reference CD4CD4 constitutive Activation/inhibition CD8 CD8a constitutiveActivation/inhibition CD8b constitutive Activation/inhibition CD3 TCRaconstitutive Activation/inhibition TCRb constitutiveActivation/inhibition CD3d constitutive Activation/inhibition CD3gconstitutive Activation/inhibition CD3e constitutiveActivation/inhibition CD3z constitutive CD3 Actin NFAT/AP1/NFkbactivated (Ca2+ dependent - CAR/TCR + CD28) CD25 NFAT/AP1/NFkb activated(Ca2+ dependent - CAR/TCR + CD28) IL2 NFAT/AP1/NFkb activated (Ca2+ 1dependent - CAR/TCR + CD28) CD69 NFAT/AP1/NFkb activated (Ca2+ 2dependent - CAR/TCR + CD28) GzmB NFAT/AP1/NFkb activated (Ca2+dependent - CAR/TCR + CD28) Th1 T-bet IFNg-IFNg-R - (STAT1) + TCR 3activation (NFAT, AP-1, NFkb) IFNg T-bet + IL2 (STAT5) TIM3 T-bet 4 Th2IL4 IL4-IL4R (STAT6) + TCR activation 5 (NFAT, AP-1, NFkb) + Th2commitment (GATA-3, c-MAF) GATA3 IL4-IL4R (STAT6) IL5 Th2 commitment(GATA-3, c-MAF) + NFAT1 IL13 Th2 commitment (GATA-3, c-MAF) + NFAT1 IL10NFAT + IRF4 6 IL27 (STAT 1/3) - IL6 (STAT3) 7 Th17 IL17A IL6-IL6R(STAT3 - ROR) + IL23- IL23R + TGFB-TGFBR IL6 IL6-IL6R (STAT3 - ROR) -TCR 8 activation (NFAT, AP-1, NFkb) IL21 IL6-IL6R (STAT3 - ROR) - TCR 9activation (NFAT, AP-1, NFkb) - IL21- IL21R IL23R IL21-IL21R -IL23-IL23R - TGFB- 10 TGFBR iTregs FoxP3 TGFB-TGFBR (SMAD), IL2/15 11(STAT5) + low affinity Antigen TCR activation (NFAT but no AP1) CTLA4NFAT + FoxP3 12 CD25 NFAT + FoxP3 PD1 NFAT/AP1/NFkb activated (Ca2+ 13dependent - CAR/TCR + CD28) 1. Jain, J., C. Loh, and A. Rao. 1995.Transcriptional regulation of the IL-2 gene. Curr. Opin. Immunol. 7:333-342. +Kim H P, Leonard W J. The basis for TCR-mediated regulation ofthe IL-2 receptor alpha chain gene: role of widely separated regulatoryelements. EMBO J 2002; 21: 3051-3059. 2. Ziegler SF, Ramsdell F,Alderson MR (1994) The activation antigen CD69. Stem Cells 12: 456-465.3. Afkarian M, Sedy JR, Yang J, et al. T-bet is a STAT1-inducedregulator of IL-12R expression in naive CD4+ T cells. Nat Immunol 2002;3: 549-557 4. Anderson A C1, Lord G M, Dardalhon V, Lee D H,Sabatos-Peyton C A, Glimcher L H, Kuchroo V K.. 2010. Th1 transcriptionfactor T-bet regulates the expression of Tim-3. Eur J Immunol. 2010March; 40(3): 859-66. doi: 10.1002/eji.200939842. 5. Chuvpilo S,Schomberg C, Gerwig R, et al. Multiple closely-linked NFAT/octamer andHMG I(Y) binding sites are part of the interleukin-4 promoter. NucleicAcids Res 1993; 21: 5694-5704. 6. Lee C G, Kang K H, So J S, et al. Adistal cis-regulatory element, CNS-9, controls NFAT1 and IRF4-mediatedIL-10 gene activation in T helper cells. Mol Immunol 2009; 46: 613-6217. Iyer S S, Cheng G. 2012. Role of Interleukin 10 TranscriptionalRegulation in Inflammation and Autoimmune Disease - Crit Rev Immunol.2012; 32(1): 23-63. 8. Macian F. NFAT proteins: key regulators of T-celldevelopment and function. Nat Rev Immunol 2005; 5: 472-484. 9. Mehta DS, Wurster A L, Weinmann A S, Grusby M J. NFATc2 and T-bet contribute toT-helper-cell-subset-specific regulation of IL-21 expression. Proc NatlAcad Sci USA 2005 10. Zhou L, Lopes J E, Chong M M, et al. TGFβ inducedFoxp3 inhibits Th17 cell differentiation by antagonizing RORgammatfunction. Nature 2008; 453: 236-240. 11. Fantini M C, Becker C,Monteleone G, Pallone F, Galle P R, Neurath M F. Cutting edge: TGFbetainduces a regulatory phenotype in CD4+ CD25− T cells through Foxp3induction and down-regulationof Smad7. J Immunol 2004; 172: 5149-5153.12. Wu Y, Borde M, Heissmeyer V, et al. Foxp3 controls regulatory T cellfunction through cooperation with NFAT. Cell 2006; 126: 375-387. 13.Oestreich K J, Yoon H, Ahmed R, Boss J M. 2008. NFATc1 Regulates PD-1Expression upon T Cell Activation1. J Immunol.; 181(7): 4832-9.

T Cell Subset-Specific Promoters. In one embodiment, a therapeutictransgene is integrated at a site within the genome of a T cell suchthat expression of the transgene is placed under control of anendogenous promoter that is active in a subset of T cells. It isunderstood that such promoters that are active in a subset of T cellsare inactive or have low activity in other T cells. Exemplary promotersthat are active in a subset of T cells include, but are not limited to,promoters such as CD4 promoter, CD8a promoter, CD8b promoter, TCRapromoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter,CD3z promoter, actin promoter, CD25 promoter, IL2 promoter, CD69promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3promoter, IL4 promoter, GATA3 promoter, IL5 promoter, IL13 promoter,IL10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23Rpromoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter,CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28promoter, CD27 promoter, CD127 promoter, PD-1 promoter, CD122 promoter,CD132 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69promoter, Ki-67 promoter, CD11a promoter, CD58 promoter, CD99 promoter,CD62L promoter, CD103 promoter, CCR4 promoter, CCR5 promoter, CCR6promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter,CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforinpromoter, CD57 promoter, CD161 promoter, IL-18Ra promoter, c-Kitpromoter, and CD130 promoter (see Tables 1 and 2).

In Table 2, the expression levels are compared to naive T cells in thedifferent T cells differentiation subsets, as reported by Mahnke et al.,Eur. J. Immunol. 43(11):2797-809. doi: 10.1002/eji.201343751 (2013).After activation by a TCR or a CAR, T cells are going throughdifferentiation, and specific genes are being activated or repressed.The inducer is the initial activation by a TCR or CAR, but signalingalso continues the co-stimulations that will impact on thedifferentiation of the T cell (see also Mahnke et al., Eur. J. Immunol.43(11):2797-809. doi: 10.1002/eji.201343751 (2013)).

TABLE 2 Exemplary Promoters Specific for T Cell Subsets (see Mahnke etal., Eur. J. Immunol. 43(11): 2797-809. doi: 10.1002/eji.201343751(2013)). stem cell central transitional effector terminal Naïve mem memmem mem eff CD45RO − − + + + − CCR7 + + + − − − CD28 + + + + − − CD95− + + + + + CD28 + ++ ++ ++ − − CD27 ++ + + + −/+ − CD127 ++ +++ +++ ++−/+ − PD-1 − −/+ + ++ + + CD122 − + ++ +++ +++ +++ CD132 + + + + + +KLRG-1 − ND −/+ + ++ +++ HLA-DR − − −/+ −/+ + − CD38 + −/+ − − − − CD69− − − − − − Ki-67 − − −/+ −/+ −/+ − CD11a + ++ ++ +++ +++ +++ CD58 − +++ +++ +++ +++ CD99 −/+ + ++ ++ ++ ++ CD62L + + + − − − CD103 − − − − +− CCR4 −/+ + ++ +++ +++ −/+ CCR5 − − + ++ +++ ++ CCR6 − − ++ +++ +++ −CCR9 CD4 − ND + − − − CD8 − ND + ++ ++ − CCR10 − − + ND ++ − CXCR3 CD4 −−/+ + ++ +++ +++ CD8 ++ +++ +++ ++ + + CXCR4 + ++ +++ +++ ++ ++ CLA −ND + ND ++ ND Granzyme A CD4 − − − − −/+ + CD8 − − −/+ ++ +++ +++Granzyme B CD4 − − − − −/+ −/+ CD8 − − − + ++ +++ Perforin CD4 − − − −−/+ −/+ CD8 − − −/+ + ++ +++ CD57 − − − −/+ ++ +++ CD161 − − −/+ + ++++++ IL-18Ra − −/+ + ++ +++ +++ c-Kit − − − ND +++ ND CD130 ++ + −/+ − −−

In general, there is usually not a single inducer for a single promoter,but signal pathways engaging and activation/repression loops that leadto promoter activation. These signaling pathways are triggered bymultiple inducers and result in the commitment of the T cells to asubset or a phenotype. However, certain genes expression patterns arevery specific to subsets and phenotypes; and their promoter can betargeted, e.g., T-bet and INFgamma in Th1; GATA3, IL4 and IL10 in Th2;IL6 in Th17; FoxP3 in Treg. Thus, an endogenous promoter can be selectedfor integration of a transgene to provide for expression of thetransgene in a particular T cell subtype.

Inducible Promoters. In one embodiment, a therapeutic transgene isintegrated at a site within the genome of a T cell such that expressionof the transgene is placed under control of an endogenous promoter thatis inducible. An inducible promoter is one that is responsive to aninducer that propagates a signal to the nucleus, resulting in activationof the inducible promoter (see, for example, Table 1). In general, theinducer is a binding partner of a molecule expressed by the T cell. Forexample, in the case of a receptor, the binding partner can be itscognate ligand, or in the case of a CAR, CCR or TCR, the binding partnercan be a target antigen.

In one embodiment, the endogenous inducible promoter is induced byactivation of the T cell. In one embodiment, the endogenous induciblepromoter is induced by binding of a chimeric antigen receptor (CAR) or achimeric co-stimulatory receptor (CCR) expressed by the T cell to itsrespective binding partner, for example, upon interaction with itscorresponding antigen. A more detailed description of CARs and CCRs areprovided under the section below describing therapeutic transgenes.Briefly, both CARs and CCRs contain intracellular signaling domains. Inthe case of a CAR, the intracellular signaling domain activates a Tcell, and optionally contains a co-stimulatory domain (in the case ofsecond and third generation CARs) (see Sadelain et al., Cancer Discov.3(4):388-398 (2013)). In the case of a CCR, it contains a co-stimulatorysignal but does not have a T cell activation signal (Sadelain et al.,supra, 2013). Binding of a corresponding antigen to a CAR or CCR resultsin activation of the T cell signaling domain and/or the co-stimulatorydomain. The activation of these signaling domains results in propagationof a signal to the nucleus and activation of certain endogenouspromoters in the T cell. Intracellular signaling domains of a CAR or CCRinclude, but are not limited to, the intracellular domains of CD28,4-1BB, CD27, ICOS, CD3z, and the like, as well as other intracellularsignaling domains disclosed herein. Signaling can also occur withmutated (e.g, mutated ITAMs), truncated or fused versions of thesedomains.

In another embodiment, the endogenous inducible promoter is induced bybinding of the T cell receptor (TCR), CD28, CD27, 4-1BB, and the like,expressed by the T cell to its respective binding partner. Thesemolecules contain intracellular signaling domains. Upon activation, thesignaling domain results in propagation of a signal to the nucleus andactivation of certain endogenous promoters in the T cell. In anotherembodiment, the endogenous inducible promoter is induced by binding ofan iCAR (CAR with inhibitory intracellular domain such as PD1, CTLA4) ortruncated CAR (no intracellular domain). In one embodiment, the iCARfunctions as a ‘break’ for the T cells activation upon target encounterthrough the signaling of CTLA4 or PD1 intracellular domains. Thuspromoters that are regulated by PD1 or CTLA4 can be used to express atransgene upon iCAR encounter with the antigen. The transgene could forexample be an immunosuppressive molecule to further control T cellactivation.

We believe that truncated CARs would allow to address the T cell to aspecific location where its target is expressed. We also believe thatthe created contact between CAR T cells and target cells wouldeventually regulate promoters than thus can be targeted for transgeneexpression

In a particular embodiment, the promoter that is induced by a CAR, CCRor TCR is selected from the group consisting of nuclear factor ofactivated T cells (NFAT) promoter, PD-1 promoter, TIM-3 promoter, CTLA4promoter, LAG3 promoter, TRAIL promoter, BTLA promoter, CD25 promoter,CD69 promoter, FasL promoter, TIGIT promoter, and 2B4 promoter. In aparticular embodiment, CAR and TCR can both regulate promoters that arein the signal pathway of CD3 ITAM phosphorylation and regulated byCa2+-dependent transcription factors (e.g., NFAT, NFkB, AP1 or CREBregulated genes such as IL2). Such promoters result in increasedexpression upon signaling from the pathway. For CAR and CCR, genesregulated upon antigen encounter depend on the domains the CAR and CCR,respectively, are composed of, e.g., a CD28 co-stimulatory domaininduces promoters activated by he PI3K pathway, while 41BBco-stimulatory domain activation induces promoters activated by the TRAFpathway. Timely regulation of promoters, in response for example toTCR/CD28 (as well as CARs composed of CD28 and CD3zeta) activation, canbe used to regulate the timing of expression of a transgene (see FIG.17; Best et al., Nat. Immunol. 14:404-413 (2013)). For example, uponactivation and memory formation of CD8+ T cells, promoters in cluster 1(12-24 hours) include, for example, CTLA4 promoter, IFNgamma promoter,Gzmb promoter, IL2ra promoter, IL2 promoter, and the like; promoters incluster 2 (12-48 hours) include, for example, CD69 promoter and Pkm2promoter, and the like; and promoters in cluster 3 (24 hours to days)include, for example, Id2 promoter, KLRg1 promoter, Cxcr3 promoter,Cxcr3r1 promoter, Itgam promoter, and the like (see also FIG. 17 foradditional exemplary promoters and Best et al., supra, 2013). Anexemplary inducible promoter is 4-1BB promoter. Another exemplaryinducible promoter is HlF1alpha, involved in the metabolic response tohypoxia.

In another embodiment, the endogenous inducible promoter is induced bybinding of a ligand to an inhibitory receptor expressed on the T cell.Exemplary inhibitory receptors include, but are not limited to, thereceptors programmed death 1 (PD-1), cytotoxic T lymphocyte antigen-4(CTLA-4), B- and T-lymphocyte attenuator (BTLA), T cell immunoglobulinmucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL, receptors 1 and2), Fas, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and 2B4(CD244). The corresponding ligands for these inhibitory receptorsinclude, for example, PD-L1 (for PD-1); PD-L2 (for PD-1); CD80, CD86(for CTLA-4); HVEM (for BTLA); Galectin-9, HMGB1 (for TIM-3); MHC II(for LAG-3); TRAIL (for TRAIL receptor 1 and TRAIL receptor 2); Fasligand (FasL) (for Fas), and the like (see Chen et al., Nat. Rev.Immunol. 13(4):227-242 (2013); Tollefson et al., J. Virol. 75:8875-8887(2001); Waring et al., Immunol. Cell Biol. 77:312-317 (1999)).

In a particular embodiment, the promoter that is induced by binding of aligand to an inhibitory receptor is selected from the group consistingof CPT1a promoter and ATGL promoter.

In another embodiment, the endogenous inducible promoter is induced bybinding of a cytokine to a cytokine receptor expressed by the T cell. Inone embodiment, the cytokine is an immunostimulatory cytokine selectedfrom the group consisting of interleukin 2 (IL2), interleukin 7 (IL7),interleukin 15 (IL15), and interleukin 21 (IL21). In another embodiment,the cytokine is an immunoinhibitory cytokine, such as interleukin 10(IL10), transforming growth factor-β (TGFβ); IL4, IL9, or Thymic stromallymphopoietin (TSLP).

In a particular embodiment, the promoter is induced by a cytokineselected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

In another embodiment, the endogenous inducible promoter is induced bycontact of a cell with a nucleic acid. In a particular embodiment, thenucleic acid is selected from the group consisting of viral DNA, viralRNA, and intracellular microRNA. Exemplary promoters that are induced bycontact of the cell with a nucleic acid include, but are not limited to,promoters of the Type I interferons (IFNs) (alpha and beta), IRF3 andIRF7 transcription factors, NFkB and AP-1 transcription factors,pro-inflammatory cytokines (TNF-alpha, IL1, IL6), and the like.

In another embodiment, the endogenous inducible promoter is induced by ametabolite. In a particular embodiment, the metabolite is selected fromthe group consisting of pyruvate, glutamine, beta-hydroxybutyrate,lactate, and serine. These metabolites are generated or taken up duringT cell activation, which translates into a metabolic change in the Tcell. Exemplary promoters that are induced by a metabolite are those of:c-Myc, HIF-1alpha, ERRalpha, CD98, SLC1A5, Psat1, Phgdh, psph, Mthfd2,Mthfd1, Mat2a, Mtrr, Mtr, Shmt1, Shmt2 (see Wang et al., Immunity35:871-882 (2011); Chang et al., Nat. Immunol. 17: 364-368 (2016); Ma etal., Cell Metab. 25:345-357 (2017)).

In another embodiment, the endogenous inducible promoter is induced by ametabolic change. This refers to the metabolic state of the cells. Forexample, when naive T cells rely on oxidative phosphorylation togenerate energy, and when they became activated and differentiate intoeffector T cell, they switch to glycolysis to generate energy. Hypoxiaand low-pH also induce metabolic changes (Chang et al., Nat. Immunol17:364-368 (2016); McNamee et al., Immunol. Res. 55: 58-70 (2013)).

In a particular embodiment, the promoter induced by a metabolic changeis PKM2 promoter. The PKM2 promoter is the same as for PKM1. PKM2 isgenerated through alternative splicing when cells switch from oxidativephosphorylation to glycolysis.

In another embodiment, the endogenous inducible promoter is induced byan ion, such as a particular ion concentration. In one embodiment, theion is potassium or calcium. Exemplary promoters induced by an ioninclude, but are not limited to the promoters of, IL2, TNFalpha, andIFNgamma, which are activated in a NFAT-dependent manner. NFAT isactivated by increased levels of intracellular calcium.

7.5 Therapeutic Transgenes

The invention relates to expressing a therapeutic transgene in a T cellby integrating the transgene at a site within the genome of the T cellsuch that expression of the transgene is under the control of anendogenous promoter of the T cell. A therapeutic transgene is anucleotide (e.g., DNA or a modified form thereof) sequence encoding atherapeutic protein or therapeutic nucleic acid. The therapeutic proteinor therapeutic nucleic acid when expressed by the T cell has use intreating a human or veterinary disease or disorder. The therapeuticnucleic acid is preferably a therapeutic RNA. The therapeutic proteincan be a peptide or polypeptide.

In one aspect of the embodiments described herein, the therapeutictransgene does not encode a membrane-bound form of interleukin 4 (IL4).

Therapeutic transgenes include, but not limited to, those encoding aCAR, chimeric co-stimulatory receptor (CCR), TRC, cytokine, dominantnegative, microenvironment modulator, antibody, biosensor, chimericreceptor ligand (CRL), chimeric immune receptor ligands (CIRL), solublereceptor, enzyme, ribozyme, genetic circuit, reporter, epigeneticmodifier, transcriptional activator or repressor, non-coding RNA, or thelike.

It is understood that a transgene can encode, for example, a cDNA, agene, miRNA or lncRNA, or the like. Additionally, the transgene can be apolycistronic message, i.e., arrayed cDNAs or arrayed miRNAs. Oneexemplary polycistronic transgene is the TCR chains. Polycistronicmessages can be engineered in the T cells to express multiple transgenesunder control of the same endogenous promoter. Thus, by knocking 3bicistronic transgenes at 3 selected loci, one could express 6 geneproducts in an engineered T cell. Thus, a number of transgenes can beexpressed in a T cell (1, 2, 3, 4, 5, 6 and so forth, as desired), eachunder control of separate endogenous promoters, or with some transgenes(i.e., polycistronic transgenes) under the control of the sameendogenous promoter. The multiple transgenes can be placed independentlyunder the control of a constitutive promoter or inducible. Thus, acombination of constitutive and/or inducible promoters can be used in aT cell to express multiple transgenes in the same cell.

In a specific embodiment, the transgene is polycistronic, e.g.,bicistronic. In a specific embodiment, the transgene is polycistronicand encodes more than one therapeutic protein or therapeutic RNA, whereexpression of both are under the control of the same endogenous promoterof the T cell. In a specific embodiment, the transgene is bicistronicand encodes two therapeutic proteins (for example, scFvs), wherein theexpression of the scFvs are both under the control of the sameendogenous promoter of the T cell.

In one embodiment, the therapeutic transgene encodes a TCR. In the caseof a transgene that is encoded on more than one polypeptide chain, thetransgene can be expressed from more than one polynucleotide, i.e., thetwo encoding nucleic acids (e.g., cDNAs) are coexpressed in a T cell.Accordingly, where a multi-subunit protein is desired to be expressed,the different polypeptide subunits can be expressed from differenttransgenes, i.e., the two encoding nucleotide sequences (e.g., cDNAsequences) are coexpressed in a T cell from different transgenesregulated by different endogenous T cell promoters. In one embodiment,the a and b chains of a TCR is expressed.

Chimeric Antigen Receptors (CARs). A chimeric antigen receptor (CAR) isan exemplary product encoded by a therapeutic transgene of theinvention. The CAR that is recombinantly expressed by a cell of theinvention has an antigen binding domain that binds to an antigen. Theantigen is associated with a disease or disorder present in the subjector desired to be prevented in the subject to which the T cell isadministered.

In specific embodiments, the CAR can be a “first generation,” “secondgeneration” or “third generation” CAR (see, for example, Sadelain etal., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev.257:127-133 (2014); Sharpe et al., Dis. Model Mech. 8(4):337-350 (2015);Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al.,Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75(2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain etal., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother.32:169-180 (2009)).

“First generation” CARs are typically composed of an extracellularantigen binding domain, for example, a single-chain variable fragment(scFv), fused to a transmembrane domain, which is fused to acytoplasmic/intracellular domain of the T cell receptor chain. “Firstgeneration” CARs typically have the intracellular domain from theCD3ζ-chain, which is the primary transmitter of signals from endogenousT cell receptors (TCRs) (see exemplary first generation CAR in FIG. 1A).“First generation” CARs can provide de novo antigen recognition andcause activation of both CD4⁺ and CD8⁺ T cells through their CD3ζ chainsignaling domain in a single fusion molecule, independent ofHLA-mediated antigen presentation. “Second-generation” CARs for use inthe invention comprise an antigen-binding domain fused to anintracellular signaling domain capable of activating T cells and aco-stimulatory domain designed to augment T cell potency and persistence(Sadelain et al., Cancer Discov. 3:388-398 (2013)). CAR design cantherefore combine antigen recognition with signal transduction, twofunctions that are physiologically borne by two separate complexes, theTCR heterodimer and the CD3 complex. “Second generation” CARs include anintracellular domain from various co-stimulatory molecules, for example,CD28, 4-1BB, ICOS, OX40, and the like, in the cytoplasmic tail of theCAR to provide additional signals to the cell (see exemplary secondgeneration CAR in FIG. 1A). “Second generation” CARs provide bothco-stimulation, for example, by CD28 or 4-1BB domains, and activation,for example, by a CD3t signaling domain. Preclinical studies haveindicated that “Second Generation” CARs can improve the anti-tumoractivity of T cells. For example, robust efficacy of “Second Generation”CAR modified T cells was demonstrated in clinical trials targeting theCD19 molecule in patients with chronic lymphoblastic leukemia (CLL) andacute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol.1(9):1577-1583 (2012)). “Third generation” CARs provide multipleco-stimulation, for example, by comprising both CD28 and 4-1BB domains,and activation, for example, by comprising a CD3ζ activation domain.

In the embodiments disclosed herein, the CARs generally comprise anextracellular antigen binding domain, a transmembrane domain and anintracellular domain, as described above, where the extracellularantigen binding domain binds to an antigen of interest, such as a cancerantigen or an antigen of an infectious pathogen, or of an autoimmunedisorder, or of a transplanted tissue. In a particular non-limitingembodiment, the extracellular antigen-binding domain is an scFv.

As disclosed herein, the methods of the invention involve administeringcells that have been engineered to express a CAR. The extracellularantigen-binding domain of a CAR is usually derived from a monoclonalantibody (mAb) or from receptors or their ligands.

The design of CARs is well known in the art (see, for example, reviewsby Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al.,Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech.8(4):337-350 (2015), and references cited therein). A CAR directed to adesired antigen can be generated using well known methods for designinga CAR, including those as described herein. A CAR, whether a first,second or third generation CAR, can be readily designed by fusing atarget antigen binding activity, for example, a cancer antigen bindingactivity, such as an scFv antibody directed to the antigen, to an immunecell signaling domain, such as a T cell receptorcytoplasmic/intracellular domain. As described above, the CAR generallyhas the structure of a cell surface receptor, with the antigen bindingactivity, such as an scFv, as at least a portion of the extracellulardomain, fused to a transmembrane domain, which is fused to anintracellular domain that has cell signaling activity in a T cell. TheCAR can include co-stimulatory molecules, as described herein. Oneskilled in the art can readily select appropriate transmembrane domains,as described herein and known in the art, and intracellular domains toprovide the desired signaling capability in the T cell.

A CAR for use in the present invention comprises an extracellular domainthat includes an antigen binding domain that binds to an antigen. In aspecific embodiment, the antigen binding domain binds to an antigen onthe target cancer cell or tissue. Such an antigen binding domain isgenerally derived from an antibody. In one embodiment, the antigenbinding domain can be an scFv or a Fab, or any suitable antigen bindingfragment of an antibody (see Sadelain et al., Cancer Discov. 3:38-398(2013)). Many antibodies or antigen binding domains derived fromantibodies that bind to an antigen, such as a cancer antigen, are knownin the art. Alternatively, such antibodies or antigen binding domainscan be produced by routine methods. Methods of generating an antibodyare well known in the art, including methods of producing a monoclonalantibody or screening a library to obtain an antigen bindingpolypeptide, including screening a library of human Fabs (Winter andHarris, Immunol. Today 14:243-246 (1993); Ward et al., Nature341:544-546 (1989); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press (1988); Hilyard et al., ProteinEngineering: A practical approach (IRL Press 1992); Borrabeck, AntibodyEngineering, 2nd ed. (Oxford University Press 1995); Huse et al.,Science 246:1275-1281 (1989)). For the CAR, the antigen binding domainderived from an antibody can be human, humanized, chimeric, CDR-grafted,and the like, as desired. For example, if a mouse monoclonal antibody isa source antibody for generating the antigen binding domain of a CAR,such an antibody can be humanized by grafting CDRs of the mouse antibodyonto a human framework (see Borrabeck, supra, 1995), which can bebeneficial for administering the CAR to a human subject. In a preferredembodiment, the antigen binding domain is an scFv. The generation ofscFvs is well known in the art (see, for example, Huston, et al., Proc.Nat. Acad. Sci. USA 85:5879-5883 (1988); Ahmad et al., Clin. Dev.Immunol. 2012: ID980250 (2012); U.S. Pat. Nos. 5,091,513, 5,132,405 and4,956,778; and U.S. Patent Publication Nos. 20050196754 and20050196754)).

With respect to obtaining an antigen binding activity, one skilled inthe art can readily obtain a suitable antigen binding activity, such asan antibody, using any of the well known methods for generating andscreening for an antibody that binds to a desired antigen, as disclosedherein, including the generation of an scFv that binds to an antigen,which is particularly useful in a CAR. In addition, a number of antigenantibodies, in particular monoclonal antibodies, such as cancer antigensor other antigens are commercially available and can also be used as asource for an antigen binding activity, such as an scFv, to generate aCAR.

Alternatively to using an antigen binding domain derived from anantibody, a CAR extracellular domain can comprise a ligand orextracellular ligand binding domain of a receptor (see Sadelain et al.,Cancer Discov. 3:388-398 (2013); Sharpe et al., Dis. Model Mech.8:337-350 (2015)). In this case, the ligand or extracellular ligandbinding domain of a receptor provides to the CAR the ability to targetthe cell expressing the CAR to the corresponding receptor or ligand. Ina specific embodiment, the ligand or extracellular ligand binding domainis selected such that the cell expressing the CAR is targeted to acancer cell or tumor (see Sadelain et al., Cancer Discov. 3:388-398(2013); Sharpe et al., Dis. Model Mech. 8:337-350 (2015), and referencescited therein). In an embodiment of the invention, the ligand orextracellular ligand binding domain is selected to bind to an antigenthat is the corresponding receptor or ligand (see Sadelain et al, CancerDiscov. 3:388-398 (2013)).

For a CAR directed to a target antigen, the antigen binding domain ofthe CAR is selected to bind to the target antigen (an antigen expressedon a target cell). Such a target antigen can be uniquely expressed on atarget cell, or the target antigen can be overexpressed in a target cellrelative to non-target cells or tissues. The target antigen to be boundby the CAR is chosen to provide targeting of the cell expressing the CARover non-target cells or tissues. In a preferred embodiment, for a CARdirected to a cancer antigen, the antigen binding domain of the CAR isselected to bind to an antigen expressed on a cancer cell. Such a cancerantigen can be uniquely expressed on a cancer cell, or the cancerantigen can be overexpressed in a cancer cell relative to noncancerouscells or tissues. The cancer antigen to be bound by the CAR is chosen toprovide targeting of the cell expressing the CAR over noncancerous cellsor tissues. In one embodiment of the methods of the invention fortreating a cancer, a T cell is designed to treat a cancer patient byexpressing in the cell a CAR that binds to a suitable cancer antigen ofthe patient's cancer, as described herein. Similarly, where a CAR isused to target an antigen of an infectious disease pathogen, or anautoimmune disorder, or of a transplanted tissue, the antigen can beuniquely expressed on the target or at the target site, or overexpressedrelative to non-target tissues or non-target sites.

The cancer antigen can be a tumor antigen. Any suitable cancer antigencan be chosen based on the type of cancer exhibited by a subject (cancerpatient) to be treated. It is understood that the selected cancerantigen is expressed in a manner such that the cancer antigen isaccessible for binding by the CAR. Generally, the cancer antigen to betargeted by a cell expressing a CAR is expressed on the cell surface ofa cancer cell. However, it is understood that any cancer antigen that isaccessible for binding to a CAR is suitable for targeting the CARexpressing cell to the cancer cell. Exemplary cancer antigens andexemplary cancers are provided below in Table 3.

TABLE 3 Targeted Cancer Antigens and Corresponding Cancer Targets.Antigen targeted Tumors investigated References B7-H3 Sarcoma andNeuroblastoma  (1) CD276 B7-H6 Ovarian and several solid cancers (2-4)Nkp30 CAIX Renal cell carcinoma  (5) Carbonic Anhydrase IX CEA Livermetastasis from Colon cancer, Colon, Pancreas,  (6-20) CarcinoembryonicAntigen Gastric and Lung cancers CSPG4 Melanoma, Mesothelioma,Glioblastoma, (21-24) Chondroitin sulfate proteoglycan-4 Osteosarcoma,Breast, Head and Neck cancers DNAM-1 Melanoma (25) DNAX AccessoryMolecule EpHA2 Glioblastoma and Lung cancer (26, 27) Ephrin type AReceptor 2 EpCAM Prostate cancer (28, 29) Epithelial Cell AdhesionMolecule ERBB family Head and Neck and Breast cancers (30, 31) ERBB2Prostate, Breast, Ovarian and Pancreatic cancers, (32-48) Glioblastoma,Meduloblastoma, Osteosarcoma, Ewing sarcoma, Neuroectodermal tumor,Desmoplastic small round cell tumor and Fibrosarcoma EGFRvIIIGlioma/Glioblastoma (49-56) Epidermal Growth Factor Receptor vIII FAPTumor associated fibroblast in Lung cancer, (27, 57-59) FibroblastAssociated Protein Mesothelioma, Breast and Pancreatic cancers FRα and βOvarian cancer (60-64) Folate Receptor GD2 Neuroblastoma, Edwingsarcoma, Melanoma (65-71) Disialoganglioside GD3 Melanoma and otherNeuroectodermal tumors (72, 73) Gp100/HLA-A2 Melanoma (74, 75) GPC3Hepatocellular carcinoma (76) Glypican 3 HERK-V Melanoma (77)MAGE-1/HLA-A1 Melanoma (78, 79) Melanoma Antigen E IL-11Rα Osteosarcoma(80) IL-13Rα2 Glioma/Glioblastoma (81-87) Medullobastoma Lewis-Y Ovarian(88) (89, 90) LMP1 Nasopharyngeal cancer (91) Latent Membrane Protein 1L1-CAM Glioblastoma, Neuroblastoma, Ovarian, Lung and Renal (92, 93)CD271 L1-Cellular Adhesion carcinoma Molecule Muc-1 Prostate and Breastcancers (43, 94-96) Mucin-1 Muc-16 Ovarian cancer (97, 98) Mucin-16 MSLNOvarian, Mesothelioma, Lung cancers  (99-107) Mesothelin N-camNeuroblastoma (108)  CD56 Neural cell-adhesion moleculel NKG2DL Ovarian(109, 110) NKG2D Ligands PSCA Prostate cancer (111-113) Prostate Stemcell Antigen PSMA Prostate (114-117) Prostate Specific Membrane AntigenROR1 Epithelial solid tumors (117, 118) Receptor tyrosine kinase-likeOrphan Receptor TAG72 Gastrointestinal, Colon and Breast cancers(119-122) Tumor Associated Glycoprotein 72 TRAIL R Various type ofcancer (123)  Trail Receptor VEGFR2 Tumor associated vasculature(124-127) Vascular Endothelial Growth Factor Receptor-2 CD166 Lungcancer (Teicher, Biochemical Pharmacology, CCR4 2014); T regs (Sugiyamaet al. 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Suitable cancer antigens include, but are not limited to, mesothelin(MSLN), prostate specific membrane antigen (PSMA), prostate stem cellantigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen(CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41,CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein2(EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesionmolecule (EpCAM), folate-binding protein (FBP), fetal acetylcholinereceptor (AChR), folate receptor-α and β (FRα and β), Ganglioside G2(GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2(HER-2/ERB2), Epidermal Growth Factor Receptor vIII (EGFRvIII), ERB3,ERB4, human telomerase reverse transcriptase (hTERT), Interleukin-13receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domainreceptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesionmolecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen familyA1, MAGE-A1), Mucin 16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands,cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4),tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growthfactor R2 (VEGF-R2), Wilms tumor protein (WT-1), type 1 tyrosine-proteinkinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-H6 (Nkp30),Chondroitin sulfate proteoglycan-4 (CSPG4), DNAX Accessory Molecule(DNAM-1), Ephrin type A Receptor 2 (EpHA2), Fibroblast AssociatedProtein (FAP), Gp100/HLA-A2, Glypican 3 (GPC3), HA-1H, HERK-V, IL-11Rα,Latent Membrane Protein 1 (LMP1), Neural cell-adhesion molecule(N-CAM/CD56), and Trail Receptor (TRAIL R). It is understood that theseor other cancer antigens can be utilized for targeting by a cancerantigen CAR.

As described above, a CAR also contains a signaling domain thatfunctions in the T cell expressing the CAR. Such a signaling domain canbe, for example, derived from CDζ or Fc receptor γ (see Sadelain et al.,Cancer Discov. 3:288-298 (2013). In general, the signaling domain willinduce persistence, trafficking and/or effector functions in thetransduced T cells, or precursor cells thereof (Sharpe et al., Dis.Model Mech. 8:337-350 (2015); Finney et al., J. Immunol. 161:2791-2797(1998); Krause et al., J. Exp. Med. 188:619-626 (1998)). In the case ofCDζ or Fc receptor γ, the signaling domain corresponds to theintracellular domain of the respective polypeptides, or a fragment ofthe intracellular domain that is sufficient for signaling. Exemplarysignaling domains are described below in more detail.

Exemplary polypeptides are described herein with reference to GenBanknumbers, GI numbers and/or SEQ ID NOS. It is understood that one skilledin the art can readily identify homologous sequences by reference tosequence sources, including but not limited to GenBank(ncbi.nlm.nih.gov/genbank/) and EMBL (embl.org/).

CD3ζ. In a non-limiting embodiment, a CAR can comprise a signalingdomain derived from a CD3ζ polypeptide, for example, a signaling domainderived from the intracellular domain of CD3ζ, which can activate orstimulate a T cell. CD3ζ comprises 3Immune-receptor-Tyrosine-based-Activation-Motifs (ITAMs), and transmitsan activation signal to the cell, for example, a cell of the lymphoidlineage such as a T cell, after antigen is bound. A CD3ζ polypeptide canhave an amino acid sequence corresponding to the sequence having GenBankNo. NP_932170 (GI:37595565; see below), or fragments thereof. In oneembodiment, the CD3ζ polypeptide has an amino acid sequence of aminoacids 52 to 164 of the CD3ζ polypeptide sequence provided below, or afragment thereof that is sufficient for signaling activity. See GenBankNP_932170 for reference to domains within CD3ζ, for example, signalpeptide, amino acids 1 to 21; extracellular domain, amino acids 22 to30; transmembrane domain, amino acids 31 to 51; intracellular domain,amino acids 52 to 164. It is understood that a “CD3ζ nucleic acidmolecule” refers to a polynucleotide encoding a CD3ζ polypeptide.

(NP_932170; SEQ ID NO: 16)   1MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD  61APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR

In certain non-limiting embodiments, an intracellular domain of a CARcan further comprise at least one co-stimulatory signaling domain. Sucha co-stimulatory signaling domain can provide increased activation of aT cell. A co-stimulatory signaling domain can be derived from a CD28polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOSpolypeptide, a DAP10 polypeptide, a 2B4 polypeptide, and the like. CARscomprising an intracellular domain that comprises a co-stimulatorysignaling region comprising 4-1BB, ICOS or DAP-10 have been describedpreviously (see U.S. Pat. No. 7,446,190, which is incorporated herein byreference, which also describes representative sequences for 4-1BB, ICOSand DAP-10). In some embodiments, the intracellular domain of a CAR cancomprise a co-stimulatory signaling region that comprises twoco-stimulatory molecules, such as CD28 and 4-1BB (see Sadelain et al.,Cancer Discov. 3(4):388-398 (2013)), or CD28 and OX40, or othercombinations of co-stimulatory ligands, as disclosed herein.

CD28. Cluster of Differentiation 28 (CD28) is a protein expressed on Tcells that provides co-stimulatory signals for T cell activation andsurvival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.In one embodiment, a CAR can comprise a co-stimulatory signaling domainderived from CD28. For example, as disclosed herein, a CAR can includeat least a portion of an intracellular/cytoplasmic domain of CD28, forexample an intracellular/cytoplasmic domain that can function as aco-stimulatory signaling domain (see FIG. 1B). A CD28 polypeptide canhave an amino acid sequence corresponding to the sequence having GenBankNo. P10747 or NP_006130 (GI:5453611), as provided below, or fragmentsthereof. If desired, CD28 sequences additional to the intracellulardomain can be included in a CAR of the invention. For example, a CAR cancomprise the transmembrane of a CD28 polypeptide. In one embodiment, aCAR can have an amino acid sequence comprising the intracellular domainof CD28 corresponding to amino acids 180 to 220 of CD28, or a fragmentthereof. In another embodiment, a CAR can have an amino acid sequencecomprising the transmembrane domain of CD28 corresponding to amino acids153 to 179, or a fragment thereof. See GenBank NP_006130 for referenceto domains within CD28, for example, signal peptide, amino acids 1 to18; extracellular domain, amino acids 19 to 152; transmembrane domain,amino acids 153 to 179; intracellular domain, amino acids 180 to 220. Itis understood that sequences of CD28 that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired. It isunderstood that a “CD28 nucleic acid molecule” refers to apolynucleotide encoding a CD28 polypeptide.

(NP_006130; SEQ ID NO: 17)   1MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD  61SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 121PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 181SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptorsuperfamily member 9, can act as a tumor necrosis factor (TNF) ligandand have stimulatory activity. In one embodiment, a CAR can comprise aco-stimulatory signaling domain derived from 4-1BB. A 4-1BB polypeptidecan have an amino acid sequence corresponding to the sequence havingGenBank No. P41273 or NP_001552 (GI:5730095) or fragments thereof In oneembodiment, a CAR can have a co-stimulatory domain comprising theintracellular domain of 4-1BB corresponding to amino acids 214 to 255,or a fragment thereof. In another embodiment, a CAR can have atransmembrane domain of 4-1BB corresponding to amino acids 187 to 213,or a fragment thereof. See GenBank NP_001552 for reference to domainswithin 4-1BB, for example, signal peptide, amino acids 1 to 17;extracellular domain, amino acids 18 to 186; transmembrane domain, aminoacids 187 to 213; intracellular domain, amino acids 214 to 255. It isunderstood that sequences of 4-1BB that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired. It isalso understood that a “4-1BB nucleic acid molecule” refers to apolynucleotide encoding a 4-1BB polypeptide.

(NP_001552; SEQ ID NO: 18)   1MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR  61TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 181PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 241CSCRFPEEEE GGCEL

OX40. OX40, also referred to as tumor necrosis factor receptorsuperfamily member 4 precursor or CD134, is a member of theTNFR-superfamily of receptors. In one embodiment, a CAR can comprise aco-stimulatory signaling domain derived from OX40. An OX40 polypeptidecan have an amino acid sequence corresponding to the sequence havingGenBank No. P43489 or NP_003318 (GI:4507579), provided below, orfragments thereof. In one embodiment, a CAR can have a co-stimulatorydomain comprising the intracellular domain of OX40 corresponding toamino acids 236 to 277, or a fragment thereof. In another embodiment, aCAR can have an amino acid sequence comprising the transmembrane domainof OX40 corresponding to amino acids 215 to 235 of OX40, or a fragmentthereof. See GenBank NP_003318 for reference to domains within OX40, forexample, signal peptide, amino acids 1 to 28; extracellular domain,amino acids 29 to 214; transmembrane domain, amino acids 215 to 235;intracellular domain, amino acids 236 to 277. It is understood thatsequences of OX40 that are shorter or longer than a specific delineateddomain can be included in a CAR, if desired. It is also understood thatan “OX40 nucleic acid molecule” refers to a polynucleotide encoding anOX40 polypeptide.

(NP_003318; SEQ ID NO: 19)   1MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ  61NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 121PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 181GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 241RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI

ICOS. Inducible T-cell costimulator precursor (ICOS), also referred toas CD278, is a CD28-superfamily costimulatory molecule that is expressedon activated T cells. In one embodiment, a CAR can comprise aco-stimulatory signaling domain derived from ICOS. An ICOS polypeptidecan have an amino acid sequence corresponding to the sequence havingGenBank No. NP_036224 (GI:15029518), provided below, or fragmentsthereof. In one embodiment, a CAR can have a co-stimulatory domaincomprising the intracellular domain of ICOS corresponding to amino acids162 to 199 of ICOS. In another embodiment, a CAR can have an amino acidsequence comprising the transmembrane domain of ICOS corresponding toamino acids 141 to 161 of ICOS, or a fragment thereof. See GenBankNP_036224 for reference to domains within ICOS, for example, signalpeptide, amino acids 1 to 20; extracellular domain, amino acids 21 to140; transmembrane domain, amino acids 141 to 161; intracellular domain,amino acids 162 to 199. It is understood that sequences of ICOS that areshorter or longer than a specific delineated domain can be included in aCAR, if desired. It is also understood that an “ICOS nucleic acidmolecule” refers to a polynucleotide encoding an ICOS polypeptide.

(NP_036224; SEQ ID NO: 20)   1MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ  61ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD HSHANYYFCN LSIFDPPPFK 121VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VVVCILGCIL ICWLTKKKYS SSVHDPNGEY 181MFMRAVNTAK KSRLTDVTL

DAP10. DAP10, also referred to as hematopoietic cell signal transducer,is a signaling subunit that associates with a large family of receptorsin hematopoietic cells. In one embodiment, a CAR can comprise aco-stimulatory domain derived from DAP10. A DAP10 polypeptide can havean amino acid sequence corresponding to the sequence having GenBank No.NP_055081.1 (GI:15826850), provided below, or fragments thereof. In oneembodiment, a CAR can have a co-stimulatory domain comprising theintracellular domain of DAP10 corresponding to amino acids 70 to 93, ora fragment thereof In another embodiment, a CAR can have a transmembranedomain of DAP10 corresponding to amino acids 49 to 69, or a fragmentthereof. See GenBank NP_055081.1 for reference to domains within DAP10,for example, signal peptide, amino acids 1 to 19; extracellular domain,amino acids 20 to 48; transmembrane domain, amino acids 49 to 69;intracellular domain, amino acids 70 to 93. It is understood thatsequences of DAP10 that are shorter or longer than a specific delineateddomain can be included in a CAR, if desired. It is also understood thata “DAP10 nucleic acid molecule” refers to a polynucleotide encoding anDAP10 polypeptide.

(NP_055081.1; SEQ ID NO: 21)  1MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA 61SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG

The extracellular domain of a CAR can be fused to a leader or a signalpeptide that directs the nascent protein into the endoplasmic reticulumand subsequent translocation to the cell surface. It is understood that,once a polypeptide containing a signal peptide is expressed at the cellsurface, the signal peptide has generally been proteolytically removedduring processing of the polypeptide in the endoplasmic reticulum andtranslocation to the cell surface. Thus, a polypeptide such as a CAR isgenerally expressed at the cell surface as a mature protein lacking thesignal peptide, whereas the precursor form of the polypeptide includesthe signal peptide. A signal peptide or leader can be essential if a CARis to be glycosylated and/or anchored in the cell membrane. The signalsequence or leader is a peptide sequence generally present at theN-terminus of newly synthesized proteins that directs their entry intothe secretory pathway. The signal peptide is covalently joined to theN-terminus of the extracellular antigen-binding domain of a CAR as afusion protein. Any suitable signal peptide, as are well known in theart, can be applied to a CAR to provide cell surface expression in a Tcell (see Gierasch Biochem. 28:923-930 (1989); von Heijne, J. Mol. Biol.184 (1):99-105 (1985)). Particularly useful signal peptides can bederived from cell surface proteins naturally expressed in the T cellthereof, including any of the signal peptides of the polypeptidesdisclosed herein. Thus, any suitable signal peptide can be utilized todirect a CAR to be expressed at the cell surface of a T cell.

In certain non-limiting embodiments, an extracellular antigen-bindingdomain of a CAR can comprise a linker sequence or peptide linkerconnecting the heavy chain variable region and light chain variableregion of the extracellular antigen-binding domain. In certainnon-limiting embodiments, a CAR can also comprise a spacer region orsequence that links the domains of the CAR to each other. For example, aspacer can be included between a signal peptide and an antigen bindingdomain, between the antigen binding domain and the transmembrane domain,between the transmembrane domain and the intracellular domain, and/orbetween domains within the intracellular domain, for example, between astimulatory domain and a co-stimulatory domain. The spacer region can beflexible enough to allow interactions of various domains with otherpolypeptides, for example, to allow the antigen binding domain to haveflexibility in orientation in order to facilitate antigen recognition.The spacer region can be, for example, the hinge region from an IgG, theCH₂CH₃ (constant) region of an immunoglobulin, and/or portions of CD3(cluster of differentiation 3) or some other sequence suitable as aspacer.

The transmembrane domain of a CAR generally comprises a hydrophobicalpha helix that spans at least a portion of the membrane. Differenttransmembrane domains result in different receptor stability. Afterantigen recognition, receptors cluster and a signal is transmitted tothe cell. In an embodiment, the transmembrane domain of a CAR can bederived from another polypeptide that is naturally expressed in the Tcell. In one embodiment, a CAR can have a transmembrane domain derivedfrom CD8, CD28, CD3, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4,BTLA, or other polypeptides expressed in the T cell having atransmembrane domain, including others as disclosed herein or that arewell known in the art. Optionally, the transmembrane domain can bederived from a polypeptide that is not naturally expressed in the Tcell, so long as the transmembrane domain can function in transducingsignal from antigen bound to the CAR to the intracellular signalingand/or co-stimulatory domains. It is understood that the portion of thepolypeptide that comprises a transmembrane domain of the polypeptide caninclude additional sequences from the polypeptide, for example,additional sequences adjacent on the N-terminal or C-terminal end of thetransmembrane domain, or other regions of the polypeptide, as desired.

CD8. Cluster of differentiation 8 (CD8) is a transmembrane glycoproteinthat serves as a co-receptor for the T cell receptor (TCR). CD8 binds toa major histocompatibility complex (MHC) molecule and is specific forthe class I MHC protein. In one embodiment, a CAR can comprise atransmembrane domain derived from CD8. A CD8 polypeptide can have anamino acid sequence corresponding to the sequence having GenBank No.NP_001139345.1 (GI:225007536), as provided below, or fragments thereof.In one embodiment, a CAR can have an amino acid sequence comprising thetransmembrane domain of CD8 corresponding to amino acids 183 to 203, orfragments thereof. See GenBank NP_001139345.1 for reference to domainswithin CD8, for example, signal peptide, amino acids 1 to 21;extracellular domain, amino acids 22 to 182; transmembrane domain aminoacids, 183 to 203; intracellular domain, amino acids 204 to 235. It isunderstood that additional sequence of CD8 beyond the transmembranedomain of amino acids 183 to 203 can be included in a CAR, if desired.It is further understood that sequences of CD8 that are shorter orlonger than a specific dilineated domain can be included in a CAR, ifdesired. It also is understood that a “CD8 nucleic acid molecule” refersto a polynucleotide encoding a CD8 polypeptide.

(NP_001139345.1; SEQ ID NO: 22)   1MALPVTALLL PLALLLHAAR PSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP  61RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN 121SIMYFSHFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA 181CDIYIWAPLA GTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYV

CD4. Cluster of differentiation 4 (CD4), also referred to as T-cellsurface glycoprotein CD4, is a glycoprotein found on the surface ofimmune cells such as T helper cells, monocytes, macrophages, anddendritic cells. In one embodiment, a CAR can comprise a transmembranedomain derived from CD4. CD4 exists in various isoforms. It isunderstood that any isoform can be selected to achieve a desiredfunction. Exemplary isoforms include isoform 1 (NP_000607.1,GI:10835167), isoform 2 (NP_001181943.1, GI:303522479), isoform 3(NP_001181944.1, GI:303522485; or NP_001181945.1, GI:303522491; orNP_001181946.1, GI:303522569), and the like. One exemplary isoformsequence, isoform 1, is provided below. In one embodiment, a CAR canhave an amino acid sequence comprising the transmembrane domain of CD4corresponding to amino acids 397 to 418, or fragments thereof. SeeGenBank NP_000607.1 for reference to domains within CD4, for example,signal peptide, amino acids 1 to 25; extracellular domain, amino acids26 to 396; transmembrane domain amino acids, 397 to 418; intracellulardomain, amino acids 419 to 458. It is understood that additionalsequence of CD4 beyond the transmembrane domain of amino acids 397 to418 can be included in a CAR, if desired. It is further understood thatsequences of CD4 that are shorter or longer than a specific dilineateddomain can be included in a CAR, if desired. It also is understood thata “CD4 nucleic acid molecule” refers to a polynucleotide encoding a CD4polypeptide.

(NP_000607.1; SEQ ID NO: 23)   1MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK  61ILGNQGSFLT KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL 121LVFGLTANSD THLLQGQSLT LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG 181TWTCTVLQNQ KKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWW 241QAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQ YAGSGNLTLA 301LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLML SLKLENKEAK VSKREKAVWV 361LNPEAGMWQC LLSDSGQVLL ESNIKVLPTW STPVQPMALI VLGGVAGLLL FIGLGIFFCV 421RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPI

It is understood that domains of the polypeptides described herein canbe used in a cancer antigen CAR, as useful to provide a desired functionsuch as a signal peptide, antigen binding domain, transmembrane domain,intracellular signaling domain and/or co-stimulatory domain. Forexample, a domain can be selected such as a signal peptide, atransmembrane domain, an intracellular signaling domain, or otherdomain, as desired, to provide a particular function to a CAR of theinvention. Possible desirable functions can include, but are not limitedto, providing a signal peptide and/or transmembrane domain.

Chimeric Co-stimulatory Receptors (CCRs). A chimeric co-stimulatoryreceptor (CCR) is an exemplary product encoded by a therapeutictransgene of the invention. Chimeric co-stimulatory receptors (CCRs) arechimeric receptors that, similar to a CAR, comprise an antigen-bindingextracellular domain, a transmembrane domain and an intracellularsignaling domain (Sadelain et al., Cancer Discov. 3(4):388-398 (2013)).CCRs do not have a T cell activation domain, but do comprise aco-stimulatory domain, such as one of the co-stimulatory domainsdescribed above for a CAR, for example, CD28, 4-1BB, OX40, ICOS, DAP10,2B4, CD70, or the like. CCRs can be used in conjunction with a T cellreceptor or a CAR to enhance T cell reactivity against dual-antigenexpressing T cells (Sadelain et al., supra, 2013). CCRs can also be usedto enhance selective tumor targeting (Sadelain et al., supra, 2013). ACCR is an antigen-specific co-stimulatory receptor, which mimics theaffects 4-1BB, OX40, ICOS or CD70 (depending on the co-stimulatorydomain of the CCR) upon binding to its binding partner, i.e., a targetantigen.

Exemplary Costimulatory Ligands (CLs) useful as a product that can beencoded by a therapeutic transgene include, but are not limited to,costimulatory ligands 4-1BBL; OX40L; ICOSL; CD70, and the like.Exemplary Chimeric Costimulatory Receptors (CCRs)that can be encoded bya therapeutic transgene include, but are not limited to, anantigen-specific costimulatory receptor, mimicking the effects of 4-1BB,OX40, ICOS or CD70 upon binding to a target antigen.

Cytokines. A cytokine is an exemplary product encoded by a therapeutictransgene of the invention. Cytokines that are particularly useful whenencoded by a therapeutic transgene include those that stimulate orsustain activation of a T cell of the invention. Exemplary cytokinesuseful as a product encoded by a therapeutic transgene for stimulatingan immune response include, but are not limited to, IL2, IL12, IL15,IL18, and the like. Exemplary cytokines useful as a product encoded by atherapeutic transgene for inhibiting an immune response include, but arenot limited to, TGFBeta, IL10, and the like.

Dominant negatives. A dominant negative is an exemplary product ofencoded by a therapeutic transgene of the invention. Dominant negativesthat are particularly useful when encoded by a therapeutic transgeneinclude those that stimulate or sustain activation of a T cell of theinvention. Exemplary dominant negatives include, but are not limited to,an inhibitory chimeric antigen receptor (iCAR), a secretable solublecytokine receptor (e.g., for TGFBeta, IL10), a secretable soluble T-cellinhibitory receptor (e.g., derived from PD1, CTLA4, LAG3, or TIM-3), andthe like. Inhibitory chimeric antigen receptors are cell-surfacereceptors composed of an extracellular scFV domain (binds a cell-surfacemolecule in the target cell) fused to an intracellular signaling domainderived from inhibitory T-cell receptors (such as PD1, CTL4). EngineeredT cells are inhibited upon interaction with a target cell.

Microenvironment Modulators. Microenvironment modulators are exemplaryproducts encoded by therapeutic transgenes of the invention. Amicroenvironment modulator refers to a molecule that modulates theactivity of cells in the vicinity of the therapeutic engineered T cell.Microenvironment modulators that are particularly useful when encoded bya therapeutic transgene include those that stimulate or sustainactivation of a T cell of the invention. Exemplary microenvironmentmodulators include, but are not limited to, heparanase, Herpes VirusEntry Mediator (HVEM), also referred to as TNFRSF14, and the like.

Antibodies. An antibody is an exemplary product encoded by a therapeutictransgene of the invention. Exemplary antibodies include, but are notlimited to, an antibody against a T-cell inhibitory ligand, such asPD1L, CD80, CD86, Galectin-9, Fas ligand, and the like.

The antibody can be expressed as an immunoglobulin, for example, an IgG,or as a Bi-specific T-cell engager (BiTE), a diabody, a duel affinityre-targeting antibody (DART), a Fab, a F(ab′), a single chain variablefragment (scFv), a nanobody, a bi-specific antibody, or the like (see,for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory (1988); Chames et al., Br. J. Pharmacol.157:220-233 (2009); Rader, Blood, 117:4403-4404 (2011)).

Biosensors. A biosensor is an exemplary product encoded by a therapeutictransgene of the invention. A biosensor is a biological molecule(protein, DNA, or RNA) that, upon ligand binding, signals to the cell toproduce a specific effect. The biosensor can be, for example, abiosensor for protein, DNA, RNA, microRNA, metabolite, ion, or the like.Exemplary biosensors include, but are not limited to, toll-like receptor(TLR), which is a biosensor for DNA, RNA, toxin, and a biosensor for anion, for example, Calcium-sensing Calmodulin (CaM)-calmodulin-bindingpeptide. The expression of specific TLRs allows the engineered T cell torespond to the presence of target molecules (such as RNA, toxin) in thecytoplasm, thus triggering a determined signal that the cell can use toactivate the expression of a therapeutic molecule. A similar strategyapplies to CaM-calmodulin binding protein (which senses intracellularcalcium). These biosensors can act as an intermediate during theproduction of a therapeutic molecule, and they do so only when it isrequired to have such an effect. For example, the biosensor can be usedto sense a state of the cell and then activate expression of anothertransgene. In a particular embodiment, or example, a biosensor can beused that specifically detect a specific HIV RNA sequence. Upon binding,the biosensor undergoes a conformational change that causes the releaseof a chimeric transcription repressor which then translocates to thenucleus and specifically inhibits HIV genome transcription.

Chimeric Receptor Ligands (CRLs). A chimeric receptor ligand (CRL) is anexemplary product encoded by a therapeutic transgene of the invention. ACRL is a cell-surface ligand that is recognized by a target-cellreceptor (such as a cytokine receptor) and that, upon interaction withits specific receptor, will trigger a T-cell response. A CRL contains anintracellular domain that will initiate a T-cell response uponinteraction with its specific receptor in the target cell.

Chimeric Immune Receptor Ligands (CIRLs). A chimeric immune receptor(CIRL) is an exemplary product encoded by a therapeutic transgene of theinvention. A CIRL is a cell-surface ligand that is recognized by animmune-cell receptor (such as a TCR or Immunoglobulin) and that, uponinteraction with its specific receptor, will trigger a T-cell response.A CIRL contains an intracellular domain that will initiate a T-cellresponse upon interaction with its specific receptor in the target cell.

Soluble Receptors. A soluble receptor is an exemplary product encoded bya therapeutic transgene of the invention. Such soluble receptors can beintracellular or extracellular. Exemplary soluble receptors include, butare not limited to, IL4R, IL10R, PD1, CTL4, TIM-3, LAG3, and the like.Such soluble receptors generally have a stimulatory effect on an immuneresponse.

Solute Transporters. A solute transporter is an exemplary productencoded by a therapeutic transgene of the invention. Exemplary solutetransporters include, but are not limited to, a glucose transporter,such as Glut1 or Glut3. Effector T cells are known to require increaseduptake of glucose to generate energy. Engineered T cells expressingglucose transporters can benefit from an increased number of glucosetransporters when T cells are in a competitive environment where tumorcells (usually in larger numbers) are consuming glucose.

Enzymes. An enzyme is an exemplary product encoded by a therapeutictransgene of the invention. An exemplary therapeutic enzyme includes,but is not limited to, PKM2. Pyruvate Kinase Muscle isozyme 2 (PKM2) isan enzyme that is needed in dividing cells, such as effector T cells,with high glycolysis rate and high demand of biosynthetic precursors.Overexpression of PKM2 helps to increase biosynthetic precursors, thusimproving proliferation of engineered T cells.

Ribozymes. A ribozyme is an exemplary product encoded by a therapeutictransgene of the invention. An exemplary ribozyme that is the product ofa therapeutic transgene includes, but is not limited to, apathogen-specific or viral-specific ribozyme that cleaves pathogen orviral genome, respectively. In the case of a viral pathogen, theribozyme can thus inhibit both virus RNA reverse transcription duringvirus entrance and virus RNA genome packaging during virus assembly. Itis readily apparent that ribozymes to various pathogens, includingtargeting viral genomes, can be expressed as the product of atherapeutic transgene. In a specific embodiment, the transgene encodes aHIV-specific ribozyme that cleaves HIV RNA genome, thus inhibiting bothvirus RNA reverse transcription during virus entrance and virus RNAgenome packaging during virus assembly.

Genetic Circuits. A genetic circuit is an exemplary product encoded by atherapeutic transgene of the invention. A genetic circuit is a set ofgene expression units that are functionally connected.

One embodiment of a genetic circuit is a constitutive transcription unitthat expresses a cell-surface ligand-specific synthetic transcriptionfactor (TF) where upon ligand binding the TF moiety is released andtranslocates to the nucleus. Then, the TF binds its cognate DNA sequencein the second transcription unit (inducible by definition), whichactivates the expression of an inhibitory ligand-specific solublereceptor that is secreted into the microenvironment to trap such aninhibitory ligand. In another embodiment, a constitutive transcriptionunit that expresses a chimeric antigen receptor (CAR) that, upontarget-cell recognition, induces the expression of a secretable tumorsuppressor (such as the lymphoma-specific soIHEVM) via a NFAT-responsiveelement; in such an embodiment, the CAR is encoded by a first transgeneunder the control of a constitutive endogenous promoter, which CAR, upontarget-cell recognition, induces the expression of a secretable tumorsuppressor (such as the lymphoma-specific soIHEVM) from a transgeneunder the control of a NFAT-responsive inducible endogenous promoter. Ina particular embodiment, a synthetic TF is expressed from onetranscription unit (a single transgene integrated at one location); thesoluble receptor is expressed from a second transcription unit (a singletransgenic expression cassette, integrated at the same or differentlocation) and that expression occurs when TF bind to this secondtranscription unit.

In a particular embodiment of a genetic circuit, a CAR-expressing T cellcontains a genetic circuit composed of HIF la-dependent andTALE-VP64-dependent transcription units. When the CAR T cell is in atumor microenvironment with low oxygen levels, HIFlalpha transcriptionfactor is activated in the T cells, binds the HIF la-dependenttranscription unit and induces the expression of a chimerictranscription factor TALE-VP64, which then binds to theTALE-VP64-dependent transcription unit and stimulates the expression ofsecretable scFvs that targets an inhibitory molecule in themicroenvironment such as PD1L or CD80. From this second transcriptionunit, a recombinant HIF1a is also expressed which will result in apositive feedback to the first transcription unit. In the foregoingspecific embodiment, expression of a first transgene is under thecontrol of an endogenous inducible promoter induced by HIF1 alphatranscription factor, expression of a second transgene is under thecontrol of an endogenous inducible promoter induced by TALE-VP64, andthe first transgene encodes TALE-VP64, and the second transgene encodesthe secretable scFv. Optionally, the expression of a third transgene,which third transgene encodes HIF1a, is under the control of a differentendogenous inducible promoter that also is induced by TALE-VP64. In oneembodiment, a transcription factor can drive expression of one ormultiple gene products, the latter occurring in the case of apolycistronic message. Examples of bicistronic transcription units: thealpha and beta chains to assemble a TCR; two scFvs in tandem, and thelike. In one embodiment, a single TALE-VP64-responsive transcriptionunit will contain both the scFv and HIF1a; this is a bicistronicconstruct: two genes will be expressed from a single promoter.

In another particular embodiment, a constitutive transcription unit thatexpresses a cell-surface CD19-specific scFV-NFAT fusion protein thatupon binding to B cells the NFAT moiety is released, it translocates tothe nucleus, and binds to its cognate DNA sequence in the secondtranscription unit, from which a chimeric immune receptor ligand isexpressed. This second gene encodes a fusion protein composed on anextracellular antigen (that is recognized by a specific immunoglobulinreceptor on the target B cell) and intracellular signaling domain(s)that activates the engineered T cell, resulting in target B-cell death.Thus, in this particular embodiment, expression of a first transgene isunder the control of an endogenous constitutive promoter, which firsttransgene encodes the cell-surface CD19-specific scFv-NFAT fusionprotein, and expression of a second transgene is under the control of anendogenous inducible promoter that is induced by binding of thecell-surface CD19-specific scFv-NFAT fusion protein to B cells, and thesecond transgene encodes a fusion protein comprising (i) anextracellular antigen (that is recognized by an immunoglobulin receptoron the target B cell), and (ii) at least one intracellular signalingdomain that activates the engineered T cell, resulting in target B-celldeath (for example, due to cytolytic activity of the activated T cell)

Epigenetic Modifiers. An epigenetic modifier is an exemplary productencoded by a therapeutic transgene of the invention. An epigeneticmodifier is a protein/enzyme that catalyzes specific modifications ofthe chromatin, either the histone proteins or DNA, at a particulargenomic location. These modifications result in specific changes of geneexpression, either activation or repression. Exemplary epigeneticmodifiers include, but are not limited to, chimeric programmablesequence-specific DNA binding domain fused to p300 acetyltransferasedomain (a histone H3 acetylase), which activates target gene expression;and chimeric programmable sequence-specific DNA binding domain fused toKRAB repressor domain (a protein that recruits heterochromatin-formingcomplexes), which represses target gene expression.

Transcriptional Activators or Repressors. A transcriptional activator orrepressor (transcription factor) is an exemplary product encoded by atherapeutic transgene of the invention. The transcriptional activator orrepressor can be naturally occurring or chimeric. In some cases, anactivator for one gene can be a repressor for another gene, or viceversa. Exemplary transcription factors that can be expressed by atherapeutic transgene include, but are not limited to, Foxp3, NFAT,HIF-1alpha, and the like.

Exemplary chimeric transcriptional activators include, but are notlimited to, fusion proteins composed of a DNA binding domain (such TAL,zinc-finger, CRISPR/deactivatedCas9) and a transactivation domain (suchVP16, VP64, p65, Rta, or combinations of them), which can be designed tospecifically activate one or more genes. Thus, in a specific embodiment,a therapeutic transgene encodes a fusion protein comprising a DNAbinding domain and a transactivation domain.

Exemplary chimeric transcriptional repressors include, but are notlimited to, fusion proteins composed of a DNA binding domain (such TAL,zinc-finger, CRISPR/deactivatedCas9) and a repressor domain (such asKRAB), which can be designed to specifically repress one or more genes.Thus, in a specific embodiment, a therapeutic transgene encodes a fusionprotein comprising a DNA binding domain and a repressor domain.

Non-coding RNA. Non-coding RNA is an exemplary product encoded by atherapeutic transgene of the invention. Exemplary non-coding RNAs(microRNAs or small interfering RNAs) include those that targetinhibitory receptor gene messenger RNAs such as PD1, TIM-3, LAG3,CTLA-4, and the like. Thus, in a specific embodiment, a therapeutictransgene encodes a non-coding RNA such as a microRNA (miRNA), smallinterfering RNA (siRNA), antisense RNA, etc. In a specific embodimentwherein stimulation of the activity of the engineered T cell is desired,the non-coding RNA can target, for example, target inhibitory receptorgene messenger RNAs such as messenger RNAs of PD1, TIM-3, LAG3, CTLA-4,or the like.

In the case where stimulating an immune response is desired, atherapeutic transgene is selected preferably to encode a product thatstimulates an immune response. Such a product that stimulates an immuneresponse can be, but is not limited to, IL12, IL15, IL18, or afunctional domains derived from any of these factors. In the casewherein inhibiting an an inhibitor of an immune response is desired, atherapeutic transgene is selected preferably to encode a product thatinhibits an inhibitor of an immune response. Such a product thatinhibits an inhibitor of an immune response, can be, but is not limitedto, an antibody specific to a ligand (e.g., PD1L, CD80, CD86, orGalectin-9) that binds a T-cell inhibitory receptor (e.g., PD1, CTLA4,LAG3, or TIM3); a soluble receptor that binds a factor such TGFbeta,TNFalpha, IL4, IL6, or IL10, thus preventing the activation of thefactor's cell-surface receptor; an antigen or functional derivativethereof that binds a specific autoimmune immune receptor on a B or Tcell to induce immunological tolerance or cell death, etc. In oneembodiment, for example, PD1L, CD80, CD86, Galectin-9 ligands are knownto inhibit T-cell activity by binding to specific receptors on T cells.The therapeutic antibodies bind the ligands -not the T-cell inhibitoryreceptors; the antibody will block the interaction between the ligandand its corresponding T-cell inhibitory receptor. Therefore, engineeredT cells that secrete these antibodies will not be inhibited by theseligands. In one embodiment, for example, TGFbeta is a cytokine that alsoinhibits T-cell activity. A therapeutic soluble receptor specific forthis cytokine will block its activity by preventing its binding to thereceptor expressed on T cells. Therefore, engineered T cells thatsecrete a TGFbeta soluble receptor will not be inhibited by thiscytokine.

In another embodiment, the T cell can optionally express a transgenethat produces a reporter. A reporter is an exemplary transgene of theinvention that can be co-expressed in a T cell with a therapeutictransgene. An example of such a transgene is truncated EGF receptor(EGFRt), which allows for both detection and elimination (i.e., canfunction as a cell suicide switch), if needed, of the therapeutic Tcell. There are specific antibodies (e.g., cetuximab) that recognizethis reporter and trigger antibody-mediated target-cell death in vivo(see U.S. Pat. No. 8,802,374)). Thus, in a specific embodiment, theengineered T cell (in which expression of a therapeutic transgene isunder the control of an endogenous promoter) further comprises areporter transgene, the reporter transgene being a transgene encoding adetectable marker (preferably cell-surface) protein, wherein expressionof the reporter transgene is under the control of an endogenous promoterof the T cell (e.g., any of the endogenous promoters describedhereinabove). In a specific embodiment, the reporter transgene does notencode IL4 or a membrane-bound form of IL4. In another specificembodiment, the reporter transgene encodes a cell suicide switch. In aspecific embodiment, the cell suicide switch is EGFRt; in such anembodiment, after administration of the engineered T cell to the subjectfor therapeutic purpose, the subject can be administered an antibodythat recognizes EGFRt and triggers cell death of the engineered T cell,to shut down the T cell activity when desired post-treatment.

7.6. Methods of Treatment

The invention also relates to methods of treating a subject with T celltherapy, wherein the subject is in need of such therapy. In embodimentswherein the T cell therapy is to promote an immune response (i.e.,treating a subject in need of a stimulated immune response), by way ofexample, the subject being treated may have cancer or an infectiousdisease, and administration of the recombinant T cells of the inventionis to treat the cancer or infectious disease, respectively. The T cellsmay be targeted to the cancer or infectious disease by virtue ofrecombinantly expressing a binding partner (e.g., a CAR or antibody orreceptor) (which may be encoded by the therapeutic transgene) to atarget antigen associated with the cancer or infectious disease, or byvirtue of being sensitized to a target antigen associated with thecancer or infectious disease. In a specific embodiment using sensitizedT cells, the T cells are sensitized to an antigen of the cancer orinfectious disease, respectively. In a preferred embodiment, theinvention also relates to methods of treating a subject with CARtherapy, wherein the subject is in need of such therapy. In embodimentswherein the CAR therapy is to promote an immune response, by way ofexample, the subject being treated may have cancer or an infectiousdisease, administration of the recombinant T cells of the invention isto treat the cancer or infectious disease, and the CAR binds to anantigen of the cancer or infectious disease pathogen, respectively. Insuch embodiments, the T cell can be CD8+, CD4+, a TSCM, a TCM, effectormemory T cell, effector T cell, Th1 cell, Th2 cell, Th9 cell, Th17 cell,Th22 cell, Tfh (follicular helper) cell, or other T cell as disclosedherein.

In embodiments wherein the T cell therapy is to suppress an immuneresponse (i.e., treating a subject in need of an inhibited immuneresponse), by way of example, the subject being treated may have anautoimmune disease or is at risk of transplant rejection, andadministration of the recombinant T cells of the invention is to treatthe autoimmune disease or to promote transplant tolerance, respectively.As another example, wherein the T cell therapy is to suppress an immuneresponse, the subject being treated may be at risk for or have graftversus host disease, and administration of the recombinant (usedinterchangeably herein with “engineered”) T cells of the invention is toprevent or reduce the graft versus host disease. The T cells may betargeted to the autoimmune disease, transplant, or graft by virtue ofrecombinantly expressing a binding partner (e.g., a CAR or antibody orreceptor) (which may be encoded by the therapeutic transgene) to atarget antigen associated with the autoimmune disease (e.g., theautoantigen), transplant, or graft, or by virtue of being sensitized toa target antigen associated with the autoimmune disease, transplant, orgraft. In a specific embodiment using sensitized T cells, the T cellsare sensitized to an antigen at the site of the autoimmune reaction orthe transplanted cells or graft (or cells derived therefrom),respectively. In such embodiments, the T cell can be a T regulatory cell(Treg). In preferred embodiments wherein CAR therapy is to suppress animmune response, by way of example, the subject being treated may havean autoimmune disease or is at risk of transplant rejection,administration of the recombinant T cells of the invention is to treatthe autoimmune disease or to promote transplant tolerance, and the CARbinds to an antigen at the site of the autoimmune reaction or thetransplanted cells, respectively. As another example, the subject beingtreated may have or be at risk of graft versus host disease (GVHD),administration of the recombinant T cells of the invention is to treator prevent or reduce the risk of GVHD, and the CAR binds to an antigenassociated with the GVHD. In such embodiments, the T cell can be a Tregulatory cell (Treg). Such autoimmune disorders include, but are notlimited to, rheumatoid arthritis, systemic lupus erythematosus, celiacsprue disease, pernicious anemia, vitiligo, scleroderma, psoriasis,inflammatory bowel disease, Hashimoto's disease, Addison's disease,Graves' disease, reactive arthritis, Sjogren's syndrome, and type 1diabetes. Transplants can be organ or tissue transplants, e.g.transplants of lung, kidney, heart, intestine, liver, and pancreas, etc.Treating or preventing GVHD can be following a hematopoietic stem celltransplant of the subject.

In one embodiment, the subject has cancer. In such an embodiment, the Tcell therapy targets the cancer. In a particular embodiment, the T cellexpresses a CAR. (Thus the therapeutic transgene encodes a CAR). In apreferred embodiment, the CAR binds to a cancer antigen. The cancerantigen is chosen to target a cancer of the subject.

The invention relates to various methods of using the T cells expressinga transgene. In a specific embodiment, the cells are administered as apopulation of cells expressing a transgene. In a preferred embodiment,the invention relates to various methods of using the T cells expressinga CAR (wherein the transgene encodes a CAR). In a specific embodimentthe cells are administered as a population of cells expressing a CAR.Optionally, the cells to be administered can be purified or enriched forthe cells of the invention.

In one embodiment, the methods of the invention are used to treatcancer. In one embodiment, the T cells express a CAR. Thus, thetransgene encodes a CAR. In one embodiment, the CAR is a cancerantigen-specific CAR.

It is understood that a method of treating cancer can include any effectthat ameliorates a sign or symptom associated with cancer. Such signs orsymptoms include, but are not limited to, reducing the number ofleukemia cells, reducing tumor burden, including inhibiting growth of atumor, slowing the growth rate of a tumor, reducing the size of a tumor,reducing the number of tumors, eliminating a tumor, all of which can bemeasured using routine tumor imaging techniques well known in the art.Other signs or symptoms associated with cancer include, but are notlimited to, fatigue, pain, weight loss, and other signs or symptomsassociated with various cancers. Thus, administration of the cells ofthe invention can reduce the number of tumor cells, reduce tumor size,and/or eradicate the tumor in the subject. The tumor can be a bloodcancer or a solid tumor. The methods of the invention can also providefor increased or lengthened survival of a subject having cancer.Additionally, methods of the invention can provide for an increasedimmune response in the subject, for example, an increased immuneresponse against the cancer.

In the methods of the invention, the T cells are administered to asubject in need of T cell therapy, for example, a subject in need oftreatment, for example, treatment of cancer, an infectious disease, anautoimmune disorder, transplant rejection, and the like as disclosedherein. In a preferred embodiment of the methods of the invention, the Tcells are administered to a subject in need of CAR therapy, for example,a subject in need of treatment, for example, treatment of cancer, aninfectious disease, an autoimmune disorder, transplant rejection, andthe like as disclosed herein. The subject can be a mammal, in particulara human. Preferably, the subject is a human. A pharmaceuticalcomposition comprising a cell of the invention is administered to asubject to elicit an immune response, with the objective of palliatingthe subject's condition. Clinical improvement comprises decreased riskor rate of progression or reduction in pathological consequences of thedisorder being treated with T cell therapy, for example, cancer. In apreferred embodiment, clinical improvement comprises decreased risk orrate of progression or reduction in pathological consequences of thedisorder being treated with CAR therapy, for example, cancer.

The subject can have an advanced form of disease, in which case thetreatment objective can include mitigation or reversal of diseaseprogression, and/or amelioration of side effects. The subjects can havea history of the condition, for which they have already been treated, inwhich case the therapeutic objective can be to decrease or delay therisk of recurrence. In the case of cancer treatment, refractory orrecurrent malignancies can be treated using the cells of the invention.Optionally, a cell of the invention can be administered for treatmentprophylactically to prevent the occurrence of a disease or condition ina subject suspected of having a predisposition to a disease orcondition, for example, based on family history and/or genetic testing.

The cells of the invention are administered to a subject, such as ahuman subject, in need of T cell therapy, for example, treatment ofcancer, an infectious disease, an autoimmune disease, transplantrejection, and the like. In a preferred embodiment, the cells of theinvention are administered to a subject, such as a human subject, inneed of CAR therapy, for example, treatment of cancer, an infectiousdisease, an autoimmune disease, transplant rejection, and the like. Inthe case of cancer, the cancer can involve a solid tumor or a bloodcancer not involving a solid tumor. Cancers to be treated using thecells of the invention comprise cancers typically responsive toimmunotherapy. Exemplary types of cancers include, but are not limitedto, carcinomas, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma,brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors,carcinoid tumors, and the like. The cancer can be a solid tumor or ablood cancer that does not form a solid tumor. In the case of a solidtumor, the tumor can be a primary tumor or a metastatic tumor.

Examples of other neoplasias or cancers that can be treated using themethods of the invention include bone cancer, intestinal cancer, livercancer, skin cancer, cancer of the head or neck, melanoma (cutaneous orintraocular malignant melanoma), renal cancer (for example, clear cellcarcinoma), throat cancer, prostate cancer (for example, hormonerefractory prostate adenocarcinoma), blood cancers (for example,leukemias, lymphomas, and myelomas), uterine cancer, rectal cancer,cancer of the anal region, bladder cancer, brain cancer, stomach cancer,testicular cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, leukemias (for example, acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease, non-Hodgkin's disease, Waldenstrom'smacroglobulinemia), cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, lymphocyticlymphoma, cancer of the kidney or ureter, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,T-cell lymphoma, environmentally induced cancers including those inducedby asbestos, heavy chain disease, and solid tumors such as sarcomas andcarcinomas, for example, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular cancer, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodenroglioma, schwannoma, meningioma, melanoma,neuroblastoma, retinoblastoma, malignant pleural disease, mesothelioma,lung cancer (for example, non-small cell lung cancer), pancreaticcancer, ovarian cancer, breast cancer (for example, metastatic breastcancer, metastatic triple-negative breast cancer), colon cancer, pleuraltumor, glioblastoma, esophageal cancer, gastric cancer, and synovialsarcoma. Solid tumors can be primary tumors or tumors in a metastaticstate.

In a specific embodiment, the cells recombinantly expressing a transgenethat are administered to the subject comprise both CD4⁻ and CD8⁺ Tcells, with the aim of generating both helper and cytotoxic T lymphocyte(CTL) responses in the subject. In a preferred embodiment wherein a CARis encoded by the transgene, the cells recombinantly expressing a CARthat are administered to the subject comprise both CD4⁺ and CD8⁺ Tcells, with the aim of generating both helper and cytotoxic T lymphocyte(CTL) responses in the subject.

In one embodiment, the invention provides a method of treating a subjectwith T cell therapy in need thereof, wherein the subject is in need ofan inhibited immune response, comprising administering T cells of theinvention that are immunoinhibitory cells. In one embodiment, thesubject has an autoimmune disease. In a particular embodiment in thecase where the T cell expresses a CAR (encoded by the transgene), theCAR binds to an autoimmune antigen of the autoimmune disorder.Autoimmune disorders include, but are not limited to, rheumatoidarthritis, systemic lupus erythematosus, celiac sprue disease,pernicious anemia, vitiligo, scleroderma, psoriasis, inflammatory boweldisease, Hashimoto's disease, Addison's disease, Graves' disease,reactive arthritis, Sjogren's syndrome, and type 1 diabetes.

In another embodiment, the subject in need of treatment with animmunoinhibitory cell has an organ transplant. In such a method, the Tcells of the invention that are immunoinhibitory are administered to thesubject to enhance immune tolerance for the transplanted organ. In aparticular embodiment in the case where the T cell expresses a CAR(encoded by the transgene), the CAR binds to an antigen of thetransplanted organ. Transplants can be transplants of lung, kidney,heart, intestine, liver, and pancreas, etc.

In another embodiment, the subject in need of treatment with animmunoinhibitory cell is in need of reducing or preventing GVHD, forexample, where the subject has had a hematopoietic stem cell transplant.In such a method, the T cells of the invention that are immunoinhibitoryare administered to the subject to enhance immune tolerance by the stemcell transplant or cells derived therefrom of antigens of the subject.In a particular embodiment in the case where the T cell expresses a CAR(encoded by the transgene), the CAR binds to an antigen of thetransplanted cells.

For treatment, the amount administered is an amount effective forproducing the desired effect. An effective amount or therapeuticallyeffective amount is an amount sufficient to provide a beneficial ordesired clinical result upon treatment. An effective amount can beprovided in a single administration or a series of administrations (oneor more doses). An effective amount can be provided in a bolus or bycontinuous perfusion. In terms of treatment, an effective amount is anamount that is sufficient to palliate, ameliorate, stabilize, reverse orslow the progression of the disease, or otherwise reduce thepathological consequences of the disease. The effective amount can bedetermined by the physician for a particular subject. Several factorsare typically taken into account when determining an appropriate dosageto achieve an effective amount. These factors include age, sex andweight of the subject, the condition being treated, the severity of thecondition and the form and effective concentration of the cells of theinvention being administered.

The cells of the invention are generally administered as a dose based oncells per kilogram (cells/kg) of body weight. Generally the cell dosesare in the range of about 10⁴ to about 10¹⁰ cells/kg of body weight, forexample, about 10⁵ to about 10⁹, about 10⁵ to about 10⁸, about 10⁵ toabout 10⁷, or about 10⁵ to 10⁶, depending on the mode and location ofadministration. In general, in the case of systemic administration, ahigher dose is used than in regional administration, where the T cellsof the invention are administered in the region, an organ or a tumor.Exemplary dose ranges include, but are not limited to, 1×10⁴ to 1×10⁸,2×10⁴ to 1×10⁸, 3×10⁴ to 1×10⁸, 4×10⁴ to 1×10⁸, 5×10⁴ to 1×10⁸, 6×10⁴,to 1×10⁸, 7×10⁴ to 1×10⁸, 8×10⁴ to 1×10⁸, 9×10⁴ to 1×10⁸, 1×10⁵ to1×10⁸, for example, 1×10⁵ to 5×10⁷, 1×10⁵ to 4×10⁷, 1×10⁵ to 3×10⁷,1×10⁵ to 2×10⁷, 1×10⁵ to 1×10⁷, 1×10⁵ to 9×10⁶, 1×10⁵ to 8×10⁶, 1×10⁵ to7×10⁶, 1×10⁵ to 6×10⁶, 1×10⁵ to 5×10⁶, 1×10⁵ to 4×10⁶, 1×10⁵ to 3×10⁶,1×10⁵ to 2×10⁶, 2×10⁵ to 7×10⁶, 2×10⁵ to 6×10⁶, 2×10⁵ to 5×10⁶, 2×10⁵ to4×10⁶, 3×10⁵ to 3×10⁶, and the like. Such dose ranges can beparticularly useful for regional administration. In a particularembodiment, cells are provided in a dose of 1×10⁵ to 5×10⁶, inparticular 1×10⁵ to 3×10⁶ or 3×10⁵ to 3×10⁶ cells/kg for regionaladministration, for example, intrapleural administration. Exemplary doseranges also can include, but are not limited to, 5×10⁵ to 1×10⁸, forexample, 6×10⁵ to 1×10⁸, 7×10⁵ to 1×10⁸, 8×10⁵ to 1×10⁸, 9×10⁵ to 1×10⁸,1×10⁶ to 1×10⁸, 1×10⁶ to 9×10⁷, 1×10⁶ to 8×10⁷, 1×10⁶ to 7×10⁷, 1×10⁶ to6×10⁷, 1×10⁶ to 5×10⁷, 1×10⁶ to 4×10⁷, 1×10⁶ to 3×10⁷, and the like.Such does can be particularly useful for systemic administration. In aparticular embodiment, cells are provided in a dose of 1×10⁶ to 3×10⁷cells/kg for systemic administration. Exemplary cell doses include, butare not limited to, a dose of 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴,7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵,8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶,9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷,1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸ ⁸ 1×10⁹ and soforth, in the range of about 10⁴ to about 10¹⁰. In addition, the dosecan also be adjusted to account for whether a single dose is beingadministered or whether multiple doses are being administered. Theprecise determination of what would be considered an effective dose canbe based on factors individual to each subject, including their size,age, sex, weight, and condition of the particular subject, as describedabove. Dosages can be readily determined by those skilled in the artbased on the disclosure herein and knowledge in the art.

The cells of the invention can be administered by any methods known inthe art, including, but not limited to, pleural administration,intravenous administration, subcutaneous administration, intranodaladministration, intratumoral administration, intrathecal administration,intrapleural administration, intraperitoneal administration,intracranial administration, and direct administration to the thymus. Inone embodiment, the cells of the invention can be delivered regionallyto an organ, a tumor or site of an autoimmune disease or site of aninfectious disease using well known methods, including but not limitedto, hepatic or aortic pump; limb, lung or liver perfusion; in the portalvein; through a venous shunt; in a cavity or in a vein that is nearby atumor, and the like. In another embodiment, the cells of the inventioncan be administered systemically. In still another embodiment, the cellsare administered regionally at the site of a desired therapy, forexample, at the site of a tumor. In the case of a tumor, the cells canalso be administered intratumorally, for example, by direct injection ofthe cells at the site of a tumor and/or into the tumor vasculature. Oneskilled in the art can select a suitable mode of administration based onthe type of target tissue or target region and/or location of a targettissue or target region to be treated. The cells can be introduced byinjection or catheter. Optionally, expansion and/or differentiationagents can be administered to the subject prior to, during or afteradministration of cells to increase production of the cells of theinvention in vivo.

Proliferation of the cells of the invention is generally done ex vivo,prior to administration to a subject, and can be desirable in vivo afteradministration to a subject (see Kaiser et al., Cancer Gene Therapy22:72-78 (2015)). Cell proliferation should be accompanied by cellsurvival to permit cell expansion and persistence, such as with T cells.Thus, the T cells can proliferate ex vivo or in vivo, as desired.

The methods of the invention can further comprise adjuvant therapy incombination with, either prior to, during, or after treatment with thecells of the invention. Thus, the cell therapy methods of the inventioncan be used with other standard care and/or therapies that arecompatible with administration of the cells of the invention.

7.7. Pharmaceutical Compositions

The invention additionally provides pharmaceutical compositionscomprising the cells of the invention. The pharmaceutical compositioncomprises an effective amount of a cell of the invention and apharmaceutically acceptable carrier. The cells of the invention andcompositions comprising the cells can be conveniently provided insterile liquid preparations, for example, typically isotonic aqueoussolutions with cell suspensions, or optionally as emulsions,dispersions, or the like, which are typically buffered to a selected pH.The compositions can comprise carriers, for example, water, saline,phosphate buffered saline, and the like, suitable for the integrity andviability of the cells, and for administration of a cell composition.

Sterile injectable solutions can be prepared by incorporating cells ofthe invention in a suitable amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionscan include a pharmaceutically acceptable carrier, diluent, or excipientsuch as sterile water, physiological saline, glucose, dextrose, or thelike, that are suitable for use with a cell composition and foradministration to a subject such as a human. Suitable buffers forproviding a cell composition are well known in the art. Any vehicle,diluent, or additive used is compatible with preserving the integrityand viability of the cells of the invention.

The compositions will generally be isotonic, that is, they have the sameosmotic pressure as blood and lacrimal fluid. The desired isotonicity ofthe cell compositions of the invention can be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, or other inorganic or organic solutes.Sodium chloride is preferred particularly for buffers containing sodiumions. One particularly useful buffer is saline, for example, normalsaline. Those skilled in the art will recognize that the components ofthe compositions should be selected to be chemically inert and will notaffect the viability or efficacy of the cells of the invention and willbe compatible for administration to a subject, such as a human. Theskilled artisan can readily determine the amount of cells and optionaladditives, vehicles, and/or carrier in compositions to be administeredin methods of the invention.

The cells of the invention can be administered in any physiologicallyacceptable vehicle. Suitable doses for administration are describedherein. A cell population comprising cells of the invention can comprisea purified population of cells. Those skilled in the art can readilydetermine the percentage of cells in a cell population using variouswell-known methods, as described herein. The ranges of purity in cellpopulations comprising genetically modified cells of the invention canbe from about 25% to about 50%, from about 30% to about 50%, from about30% to about 40%, from about 40% to 50%, from about 50% to about 55%,from about 55% to about 60%, from about 65% to about 70%, from about 70%to about 75%, from about 75% to about 80%, from about 80% to about 85%;from about 85% to about 90%, from about 90% to about 95%, or from about95 to about 100%. It is understood that such a population can begenerated efficiently with the methods of the invention, as disclosedherein, or optionally enriched for the genetically modified cellsexpressing a transgene, as disclosed herein. In a preferred embodimentwherein the transgene encodes a CAR, it is understood that such apopulation can be generated efficiently with the methods of theinvention, as disclosed herein, or optionally enriched for thegenetically modified cells expressing a CAR, as disclosed herein.Dosages can be readily adjusted by those skilled in the art; forexample, a decrease in purity may require an increase in dosage.

The invention also provides kits for preparation of cells of theinvention. In one embodiment, the kit comprises in one or morecontainers: one or more vectors for generating a genetically engineeredT cell that contains a transgene integrated within its genome such thatexpression of the transgene is under control of an endogenous promoterof the T cell. In a preferred embodiment, the transgene is a CAR, and ina particular embodiment, the kit comprises one or more vectors forgenerating a genetically engineered T cell that expresses a CAR. In aparticular embodiment, the kit comprises in a container a recombinantnon-integrating gamma retrovirus, as disclosed herein. The kit can alsocontain a suitable homologous recombination system, such as azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN),a clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, Meganuclease or aMega-Tal, preferably in a separate container. The kits can be used togenerate genetically engineered T cells from autologous cells derivedfrom a subject or from non-autologous cells to be administered to acompatible subject. In another embodiment, the kits can comprise cellsof the invention for autologous or non-autologous administration to asubject. In specific embodiments, the kit comprises the T cells of theinvention in one or more containers.

In another embodiment, the invention provides a kit comprising arecombinant non-integrating gamma retrovirus, as disclosed herein. Inspecific embodiments, the kit comprises the non-integrating gammaretrovirus of the invention in one or more containers.

7.8. Alternative Embodiment Relating to Reporter Transgenes

In an alternative specific embodiment of the invention, a T cell hasintegrated into its genome a reporter transgene (optionally instead of atherapeutic transgene), wherein the expression of the reporter transgeneis under the control of an endogenous promoter, which can be any of thepromoters described hereinabove. The reporter transgene is a transgeneencoding a detectable marker (preferably cell-surface) protein. In aspecific embodiment, the reporter transgene does not encode IL4 or amembrane-bound form of IL4. In another specific embodiment, the reportertransgene encodes a cell suicide switch. In a specific embodiment, thecell suicide switch is a truncated EGF receptor (EGFRt), which allowsfor both detection and elimination, if needed, of the therapeutic Tcell. There are specific antibodies (e.g., cetuximab) that recognizethis reporter and trigger antibody-mediated target-cell death in vivo(see U.S. Pat. No. 8,802,374). Thus, for example, the transgene encodingthe EGFRt can under the control of an endogenous constitutive orinducible promoter, and after administration of the engineered T cell tothe subject for therapeutic purpose, the subject can be administered anantibody that recognizes EGFRt and triggers cell death of the engineeredT cell, to shut down the T cell activity when desired post-treatment.

7.9. Exemplary Embodiments

The invention provides the following exemplary embodiments.

Embodiment 1. A T cell wherein a transgene is integrated at a first sitewithin the genome of the T cell such that expression of the transgene isunder control of an endogenous promoter of the T cell, wherein thetransgene encodes a therapeutic protein or therapeutic nucleic acid.

Embodiment 2. The T cell of embodiment 1, wherein the transgene encodesa therapeutic protein.

Embodiment 3. The T cell of embodiment 1, wherein the transgene encodesa therapeutic nucleic acid.

Embodiment 4. The T cell of any one of embodiments 1-3, wherein thetransgene is integrated at a single site within the genome.

Embodiment 5. The T cell of any one of embodiments 1-3, wherein thetransgene is integrated at two sites within the genome of the cell.

Embodiment 6. The T cell of any one of embodiments 1-5, wherein thefirst site is an exon of the endogenous gene under control of theendogenous promoter.

Embodiment 7. The T cell of embodiment 6, wherein the first site iswithin the first exon of the endogenous gene.

Embodiment 8. The T cell of any one of embodiments 1-7, wherein theendogenous promoter is constitutive.

Embodiment 9. The T cell of embodiment 8, wherein the promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

Embodiment 10. The T cell of any one of embodiments 1-7, wherein theendogenous promoter is active in a subset of T cells.

Embodiment 11. The T cell of embodiment 10, wherein the endogenouspromoter is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, CD3z promoter, actin promoter, CD25promoter, IL2 promoter, CD69 promoter, GzmB promoter, T-bet promoter,IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter,IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter,CD95 promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1promoter, CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DRpromoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter,CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter,CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter,Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter,IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

Embodiment 12. The T cell of any one of embodiments 1-7, wherein theendogenous promoter is inducible.

Embodiment 13. The T cell of embodiment 12, wherein the endogenouspromoter is induced by activation of the T cell.

Embodiment 14. The T cell of embodiment 12, wherein the promoter isinduced by binding of a chimeric antigen receptor (CAR), a chimericco-stimulatory receptor (CCR), T cell receptor (TCR), CD28, CD27, or4-1BB expressed by the T cell to its respective binding partner.

Embodiment 15. The T cell of embodiment 14, wherein the promoter isinduced by binding of a CAR, CCR or TCR expressed by the T cell to itsrespective binding partner.

Embodiment 16. The T cell of embodiment 15, wherein the promoter isselected from the group consisting of nuclear factor of activated Tcells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cellimmunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyteantigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3)promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and2B4 promoter.

Embodiment 17. The T cell of embodiment 12, wherein the promoter isinduced by binding of a ligand to an inhibitory receptor expressed bythe T cell.

Embodiment 18. The T cell of embodiment 17, wherein the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4.

Embodiment 19. The T cell of embodiment 17, wherein the promoter isselected from the group consisting of CPT1a promoter and ATGL promoter.

Embodiment 20. The T cell of embodiment 12, wherein the promoter isinduced by binding of a cytokine to a cytokine receptor expressed by theT cell.

Embodiment 21. The T cell of embodiment 20, wherein the cytokine isselected from the group consisting of interleukin 2 (IL2), interleukin 7(IL7), interleukin 15 (IL15), and interleukin 21 (IL21).

Embodiment 22. The T cell of embodiment 20, wherein the cytokine isselected from the group consisting of interleukin 10 (IL10) andtransforming growth factor β (TGFβ).

Embodiment 23. The T cell of embodiment 20, wherein the promoter isselected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

Embodiment 24. The T cell of embodiment 12, wherein the promoter isinduced by contact of the cell with a nucleic acid.

Embodiment 25. The T cell of embodiment 24, wherein the nucleic acid isselected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA.

Embodiment 26. The T cell of embodiment 25, wherein the promoter isselected from the group consisting of Type I interferon (IFN) alpha,Type I IFN beta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL 1, and IL6.

Embodiment 27. The T cell of embodiment 12, wherein the promoter isinduced by contact of the cell with a metabolite.

Embodiment 28. The T cell of embodiment 27, wherein the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

Embodiment 29. The T cell of embodiment 12, wherein the promoter isinduced by a metabolic change in the cell or contact of the cell with asubstance that causes a metabolic change in the cell.

Embodiment 30. The T cell of embodiment 29, wherein the promoter is PKM2promoter.

Embodiment 31. The T cell of embodiment 12, wherein the promoter isinduced by a particular ion concentration in the cell or contact of thecell with a particular ion concentration.

Embodiment 32. The T cell of embodiment 31, wherein the ion is potassiumor calcium.

Embodiment 33. The T cell of embodiment 31, wherein the promoter isselected from the group consisting of IL2 promoter, TNFalpha promoter,and IFNgamma promoter.

Embodiment 34. The T cell of any one of embodiments 1-33, wherein thetransgene encodes a molecule selected from the group consisting of aCAR, a CCR, a cytokine, a dominant negative, a microenvironmentmodulator, an antibody, a biosensor, a chimeric receptor ligand (CRL), achimeric immune receptor ligand (CIRL), a soluble receptor, a solutetransporter, an enzyme, a ribozyme, a genetic circuit, an epigeneticmodifier, a transcriptional activator, a transcriptional repressor, andnon-coding RNA.

Embodiment 35. The T cell of embodiment 34, wherein the transgeneencodes a cytokine, and optionally the cytokine is immunostimulatory.

Embodiment 36. The T cell of embodiment 35, wherein the cytokine isimmunostimulatory, and the cytokine is selected from the groupconsisting of IL2, IL12, IL15, and IL18.

Embodiment 37. The T cell of embodiment 34, wherein the transgeneencodes a cytokine, and optionally the cytokine is immunoinhibitory.

Embodiment 38. The T cell of embodiment 37, wherein the cytokine isimmunoinhibitory, and the cytokine is selected from the group consistingof TGFBeta and IL10.

Embodiment 39. The T cell of embodiment 34, wherein the transgeneencodes an antibody, and optionally the antibody is selected from thegroup consisting of an immunoglobulin, a Bi-specific T-cell engager(BiTE), a diabody, a dual affinity re-targeting (DART), a Fab, a F(ab′),a single chain variable fragment (scFv), and a nanobody.

Embodiment 40. The T cell of embodiment 34, wherein the transgeneencodes a CAR.

Embodiment 41. The T cell of embodiment 40, wherein the CAR binds to acancer antigen.

Embodiment 42. The T cell of any one of embodiments 1-39, wherein the Tcell is sensitized to a target antigen.

Embodiment 43. The T cell of any one of embodiments 1-42, wherein atransgene (hereinafter “reporter transgene”) encoding a reportermolecule is integrated within the genome of the T cell such thatexpression of the reporter transgene is under control of a promoter,preferably an endogenous promoter of the T cell.

Embodiment 44. The T cell of any one of embodiments 1-43 which isderived from a human.

Embodiment 45. The T cell of embodiment 44, wherein the T cell is aprimary human T cell, a T cell derived from a CD34 hematopoietic stemcell, a T cell derived from an embryonic stem cell, or a T cell derivedfrom an induced pluripotent stem cell.

Embodiment 46. The T cell of any one of embodiments 1-45, wherein thetransgene is integrated into the first site by targeted homologousrecombination.

Embodiment 47. The T cell of embodiment 46, wherein the targetedhomologous recombination is carried out by a method comprising using azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), a clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, pyrogen, Aureus,Meganuclease or a Mega-Tal.

Embodiment 48. The T cell of any one of embodiments 1-47, wherein thetransgene is integrated at a plurality of sites within the genome of theT cell, and such that expression of the transgene at said plurality ofsites is under the control of different endogenous promoters.

Embodiment 49. A T cell wherein a first transgene is integrated at afirst site within the genome of the cell such that expression of thefirst transgene is under control of a first endogenous promoter of the Tcell, and wherein a second transgene is integrated at a second sitewithin the genome of the cell, such that expression of the secondtransgene is under the control of a second endogenous promoter, whereinsaid first and second endogenous promoters are different promoters, andwherein the first transgene encodes a first therapeutic protein or firsttherapeutic nucleic acid, and the second transgene encodes a secondtherapeutic protein or second therapeutic nucleic acid, preferablywherein the first therapeutic protein or first therapeutic nucleic acidis different from said second therapeutic protein or second therapeuticnucleic nucleic, respectively.

Embodiment 50. The T cell of embodiment 49, wherein the first transgeneencodes a first therapeutic protein.

Embodiment 51. The T cell of embodiment 49, wherein the first transgeneencodes a first therapeutic nucleic acid.

Embodiment 52. The T cell of embodiment 49, wherein the second transgeneencodes a second therapeutic protein.

Embodiment 53. The T cell of embodiment 49, wherein the second transgeneencodes a second therapeutic nucleic acid.

Embodiment 54. The T cell of any one of embodiments 49-53, wherein thefirst endogenous promoter is constitutive, and the second endogenouspromoter is inducible.

Embodiment 55. The T cell of embodiment 54, wherein the constitutivepromoter is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, and CD3z promoter.

Embodiment 56. The T cell of embodiment 54, wherein the first endogenouspromoter and/or the second endogenous promoter is active in a subset ofT cells.

Embodiment 57. The T cell of embodiment 56, wherein the first endogenouspromoter and/or the second endogenous promoter is independently selectedfrom the group consisting of CD4 promoter, CD8a promoter, CD8b promoter,TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3epromoter, CD3z promoter, actin promoter, CD25 promoter, IL2 promoter,CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3promoter, IL4 promoter, GATA3 promoter, IL5 promoter, IL13 promoter,IL10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23Rpromoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter,CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28promoter, CD27 promoter, CD127 promoter, PD-1 promoter, CD122 promoter,CD132 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69promoter, Ki-67 promoter, CD11a promoter, CD58 promoter, CD99 promoter,CD62L promoter, CD103 promoter, CCR4 promoter, CCR5 promoter, CCR6promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter,CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforinpromoter, CD57 promoter, CD161 promoter, IL-18Ra promoter, c-Kitpromoter, and CD130 promoter.

Embodiment 58. The T cell of embodiment 54, wherein the induciblepromoter is induced by activation of the T cell.

Embodiment 59. The T cell of embodiment 54, wherein the induciblepromoter is induced by binding of a chimeric antigen receptor (CAR), achimeric co-stimulatory receptor (CCR), T cell receptor (TCR), CD28,CD27, and 4-1BB expressed by the T cell to its respective bindingpartner.

Embodiment 60. The T cell of embodiment 59, wherein the induciblepromoter is induced by binding of a CAR, CCR or TCR expressed by the Tcell to its respective binding partner.

Embodiment 61. The T cell of embodiment 60, wherein the induciblepromoter is selected from the group consisting of nuclear factor ofactivated T cells (NFAT) promoter, programmed death 1 (PD-1) promoter, Tcell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyteantigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3)promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and2B4 promoter.

Embodiment 62. The T cell of embodiment 54, wherein the induciblepromoter is induced by binding of a ligand to an inhibitory receptorexpressed by the T cell.

Embodiment 63. The T cell of embodiment 62, wherein the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4.

Embodiment 64. The T cell of embodiment 62, wherein the induciblepromoter is selected from the group consisting of CPT1a promoter andATGL promoter.

Embodiment 65. The T cell of embodiment 54, wherein the induciblepromoter is induced by binding of a cytokine to a cytokine receptorexpressed by the T cell.

Embodiment 66. The T cell of embodiment 65, wherein the cytokine isselected from the group consisting of interleukin 2 (IL2), interleukin 7(IL7), interleukin 15 (IL15), and interleukin 21 (IL21).

Embodiment 67. The T cell of embodiment 65, wherein the cytokine isselected from the group consisting of interleukin 10 (IL10) andtransforming growth factor β (TGFβ).

Embodiment 68. The T cell of embodiment 65, wherein the induciblepromoter is selected from the group consisting of T-bet promoter, Eomespromoter, GATA3 promoter, and CD45RA promoter.

Embodiment 69. The T cell of embodiment 54, wherein the induciblepromoter is induced by contact of the cell with a nucleic acid.

Embodiment 70. The T cell of embodiment 69, wherein the nucleic acid isselected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA.

Embodiment 71. The T cell of embodiment 70, wherein the induciblepromoter is selected from the group consisting of Type I interferon(IFN) alpha, Type I IFN beta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1,and IL6.

Embodiment 72. The T cell of embodiment 54, wherein the induciblepromoter is induced by contact of the cell with a metabolite.

Embodiment 73. The T cell of embodiment 72, wherein the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

Embodiment 74. The T cell of embodiment 54, wherein the induciblepromoter is induced by a metabolic change in the cell or contact of thecell with a substance that causes a metabolic change in the cell.

Embodiment 75. The T cell of embodiment 74, wherein the induciblepromoter is PKM2 promoter.

Embodiment 76. The T cell of embodiment 54, wherein the induciblepromoter is induced by a particular ion concentration in the cell orcontact of the cell with a particular ion concentration.

Embodiment 77. The T cell of embodiment 76, wherein the ion is potassiumor calcium.

Embodiment 78. The T cell of embodiment 76, wherein the induciblepromoter is selected from the group consisting of IL2 promoter, TNFalphapromoter, and IFNgamma promoter.

Embodiment 79. The T cell of any one of embodiments 49-78, wherein thefirst transgene and/or second transgene each encodes a moleculeindependently selected from the group consisting of a CAR, a CCR, acytokine, a dominant negative, a microenvironment modulator, anantibody, a biosensor, a chimeric receptor ligand (CRL), a chimericimmune receptor ligand (CIRL), a soluble receptor, a solute transporter,an enzyme, a ribozyme, a genetic circuit, an epigenetic modifier, atranscriptional activator, a transcriptional repressor, and non-codingRNA.

Embodiment 80. The T cell of embodiment 79, wherein the first transgeneand/or second transgene encodes a cytokine, preferably wherein thecytokine is immunostimulatory.

Embodiment 81. The T cell of embodiment 80, wherein the cytokine isimmunostimulatory, and is selected from the group consisting of IL2,IL12, IL15, and IL18.

Embodiment 82. The T cell of embodiment 79, wherein wherein the firsttransgene and/or second transgene encodes a cytokine, preferably whereinthe cytokine is immunoinhibitory.

Embodiment 83. The T cell of embodiment 82, wherein the cytokine isimmunoinhibitory, and is selected from the group consisting of TGFBetaand IL10.

Embodiment 84. The T cell of embodiment 79, wherein the first transgeneand/or second transgene encodes an antibody, and the antibody is animmunoglobulin, a Bi-specific T-cell engager (BiTE), a diabody, a dualaffinity re-targeting (DART), a Fab, a F(ab′), a single chain variablefragment (scFv), and a nanobody.

Embodiment 85. The T cell of embodiment 79, wherein the first transgeneand/or second transgene encodes a CAR.

Embodiment 86. The T cell of embodiment 85, wherein the CAR binds to acancer antigen.

Embodiment 87. The T cell of any one of embodiments 49-84, wherein the Tcell is sensitized to a target antigen.

Embodiment 88. The T cell of any one of embodiments 49-87, wherein atransgene (hereinafter “reporter transgene”) encoding a reportermolecule is integrated within the genome of the T cell such thatexpression of the reporter transgene is under control of a promoter,preferably an endogenous promoter of the T cell.

Embodiment 89. The T cell of any one of embodiments 49-88 which isderived from a human.

Embodiment 90. The T cell of embodiment 89, wherein the T cell is aprimary human T cell, a T cell derived from a CD34 hematopoietic stemcell, a T cell derived from an embryonic stem cell, or a T cell derivedfrom an induced pluripotent stem cell.

Embodiment 91. The T cell of any one of embodiments 49-90, wherein thefirst transgene and/or second transgene is integrated into the firstsite by targeted homologous recombination.

Embodiment 92. The T cell of embodiment 91, wherein the targetedhomologous recombination is carried out by a method comprising using azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), a clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, pyrogen, Aureus,Meganuclease or a Mega-Tal.

Embodiment 93. The T cell of any one of embodiments 49-92, wherein thefirst therapeutic protein or first therapeutic nucleic acid is differentfrom said second therapeutic protein or second therapeutic nucleicnucleic, respectively.

Embodiment 94. The T cell of embodiment 54, wherein the secondendogenous promoter is induced by activation of the T cell.

Embodiment 95. The T cell of embodiment 54, wherein the first transgeneencodes a CAR.

Embodiment 96. The T cell of embodiment 95, wherein the first endogenouspromoter is a T cell receptor promoter.

Embodiment 97. The T cell of embodiment 96, wherein the promoter isselected from the group consisting of T cell receptor alpha chainpromoter, T cell receptor beta chain promoter, CD3 gamma chain promoter,CD3 delta chain promoter, CD3 epsilon chain promoter, and CD3 zeta chainpromoter.

Embodiment 98. The T cell of embodiment 97, wherein the promoter is Tcell receptor alpha chain promoter.

Embodiment 99. The T cell of any one of embodiments 1-36, 39-81 or 84-98(except insofar as the foregoing embodiments depend directly orindirectly on embodiments 37-38), wherein the T cell is animmunostimulatory T cell.

Embodiment 100. The T cell of embodiment 99, wherein the T cell isselected from the group consisting of cytotoxic T lymphocyte (CTL), CD4+subtype, CD8+ subtype, central memory T cell (TCM), stem memory T cell(TSCM), effector memory T cell, effector T cell, Th1 cell, Th2 cell, Th9cell, Th17 cell, Th22 cell, and Tfh (follicular helper) cell.

Embodiment 101. The T cell of embodiment 100, wherein the T cell isCD4+.

Embodiment 102. The T cell of embodiment 100, wherein the T cell isCD8+.

Embodiment 103. The T cell of any one of embodiments 1-34, 37-79 or82-98 (except insofar as the foregoing embodiments depend directly orindirectly on embodiments 35-36), wherein the T cell is animmunoinhibitory T cell.

Embodiment 104. The T cell of embodiment 103, wherein the T cell is aregulatory T cell.

Embodiment 105. An isolated population of T cells, which comprises aplurality of the T cell of any one of embodiments 1-98.

Embodiment 106. An isolated population of T cells, which comprises aplurality of the T cell of any one of embodiments 99-102.

Embodiment 107. An isolated population of T cells, which comprises aplurality of the T cell of embodiment 103 or 104.

Embodiment 108. A pharmaceutical composition comprising atherapeutically effective amount of the T cell of any one of embodiments1-98; and a pharmaceutically acceptable carrier.

Embodiment 109. A pharmaceutical composition comprising atherapeutically effective amount of a population of T cells, whichpopulation comprises a plurality of the T cell of any one of embodiments1-98; and a pharmaceutically acceptable carrier.

Embodiment 110. A pharmaceutical composition comprising atherapeutically effective amount of the T cell of any one of embodiments99-102; and a pharmaceutically acceptable carrier.

Embodiment 111. A pharmaceutical composition comprising atherapeutically effective amount of a population of T cells, whichpopulation comprises a plurality of the T cell of any one of embodiments99-102 and a pharmaceutically acceptable carrier.

Embodiment 112. A pharmaceutical composition comprising atherapeutically effective amount of the T cell of embodiment 103 or 104;and a pharmaceutically acceptable carrier.

Embodiment 113. A pharmaceutical composition comprising atherapeutically effective amount of a population of T cells, whichpopulation comprises a plurality of the T cell of embodiment 103 or 104;and a pharmaceutically acceptable carrier.

Embodiment 114. A method of treating a subject with T cell therapy inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of the T cell of any one of embodiments 1-98.

Embodiment 115. A method of treating a subject with T cell therapy inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of the T cell population of embodiment 105.

Embodiment 116. A method of treating a subject with T cell therapy inneed thereof, comprising administering to the subject the pharmaceuticalcomposition of embodiment 108 or 109.

Embodiment 117. The method of any one of embodiments 114-116, whereinthe subject is a human, and wherein the cell is derived from a human.

Embodiment 118. The method of any one of embodiments 114-117, whereinthe T cell is autologous to the subject.

Embodiment 119. The method of any one of embodiments 114-117, whereinthe T cell is non-autologous to the subject.

Embodiment 120. A method of treating a subject with T cell therapy inneed thereof, wherein the subject is in need of a stimulated immuneresponse, comprising administering to the subject a therapeuticallyeffective amount of a T cell, wherein a transgene is integrated at afirst site within the genome of the T cell such that expression of thetransgene is under control of an endogenous promoter of the T cell,wherein the transgene encodes a therapeutic protein or therapeuticnucleic acid.

Embodiment 121. The method of embodiment 116, wherein the cell or cellpopulation is administered to the subject as a pharmaceuticalcomposition.

Embodiment 122. The method of embodiment 120, wherein the transgeneencodes a therapeutic protein.

Embodiment 123. The method of embodiment 120, wherein the transgeneencodes a therapeutic nucleic acid.

Embodiment 124. The method of any one of embodiments 116-123, whereinthe transgene is integrated at a single site within the genome.

Embodiment 125. The method of any one of embodiments 116-123, whereinthe transgene is integrated at two sites within the genome of the cell.

Embodiment 126. The method of any one of embodiments 116-125, whereinthe first site is an an exon of the endogenous gene under control of theendogenous promoter.

Embodiment 127. The method of embodiment 126, wherein the first site iswithin the first exon of the endogenous gene.

Embodiment 128. The method of any one of embodiments 116-127, whereinthe endogenous promoter is constitutive.

Embodiment 129. The method of embodiment 128, wherein the promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

Embodiment 130. The method of any one of embodiments 116-127, whereinthe endogenous promoter is active in a subset of T cells.

Embodiment 131. The method of embodiment 130, wherein the endogenouspromoter is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, CD3z promoter, actin promoter, CD25promoter, IL2 promoter, CD69 promoter, GzmB promoter, T-bet promoter,IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter,IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter,CD95 promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1promoter, CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DRpromoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter,CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter,CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter,Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter,IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

Embodiment 132. The method of any one of embodiments 116-127, whereinthe endogenous promoter is inducible.

Embodiment 133. The method of embodiment 132, wherein the endogenouspromoter is induced by activation of the T cell.

Embodiment 134. The method of embodiment 132, wherein the promoter isinduced by binding of a chimeric antigen receptor (CAR), a chimericco-stimulatory receptor (CCR), T cell receptor (TCR), CD28, CD27, or4-1BB expressed by the T cell to its respective binding partner.

Embodiment 135. The method of embodiment 134, wherein the promoter isinduced by binding of a CAR, CCR or TCR expressed by the T cell to itsrespective binding partner.

Embodiment 136. The method of embodiment 135, wherein the promoter isselected from the group consisting of nuclear factor of activated Tcells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cellimmunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyteantigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3)promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and2B4 promoter.

Embodiment 137. The method of embodiment 132, wherein the promoter isinduced by binding of a ligand to an inhibitory receptor expressed bythe T cell.

Embodiment 138. The nethod of embodiment 137, wherein the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4.

Embodiment 139. The method of embodiment 137, wherein the promoter isselected from the group consisting of CPT1a promoter and ATGL promoter.

Embodiment 140. The method of embodiment 132, wherein the promoter isinduced by binding of a cytokine to a cytokine receptor expressed by theT cell.

Embodiment 141. The method of embodiment 140, wherein the cytokine isselected from the group consisting of interleukin 2 (IL2), interleukin 7(IL7), interleukin 15 (IL15), and interleukin 21 (IL21).

Embodiment 142. The method of embodiment 140, wherein the promoter isselected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

Embodiment 143. The method of embodiment 132, wherein the promoter isinduced by contact of the cell with a nucleic acid.

Embodiment 144. The method of embodiment 143, wherein the nucleic acidis selected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA.

Embodiment 145. The method of embodiment 144, wherein the promoter isselected from the group consisting of Type I interferon (IFN) alpha,Type I IFN beta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1, and IL6.

Embodiment 146. The method of embodiment 132, wherein the promoter isinduced by contact of the cell with a metabolite.

Embodiment 147. The method of embodiment 146, wherein the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

Embodiment 148. The method of embodiment 132, wherein the promoter isinduced by a metabolic change in the cell or contact of the cell with asubstance that causes a metabolic change in the cell.

Embodiment 149. The method of embodiment 148, wherein the promoter isPKM2 promoter.

Embodiment 150. The method of embodiment 132, wherein the promoter isinduced by a particular ion concentration in the cell or contact of thecell with a particular ion concentration.

Embodiment 151. The method of embodiment 150, wherein the ion ispotassium or calcium.

Embodiment 152. The method of embodiment 150, wherein the promoter isselected from the group consisting of IL2 promoter, TNFalpha promoter,and IFNgamma promoter.

Embodiment 153. The method of any one of embodiments 120-152, whereinthe transgene encodes a molecule selected from the group consisting of aCAR, a CCR, a cytokine, a dominant negative, a microenvironmentmodulator, an antibody, a biosensor, a chimeric receptor ligand (CRL), achimeric immune receptor ligand (CIRL), a soluble receptor, a solutetransporter, an enzyme, a ribozyme, a genetic circuit, an epigeneticmodifier, a transcriptional activator, a transcriptional repressor, andnon-coding RNA.

Embodiment 154. The method of embodiment 153, wherein the transgeneencodes a cytokine, and optionally the cytokine is immunostimulatory.

Embodiment 155. The method of embodiment 154, wherein the cytokine isimmunostimulatory, and the cytokine is selected from the groupconsisting of IL2, IL12, IL15, and IL18.

Embodiment 156. The method of embodiment 153, wherein the transgeneencodes an antibody, and optionally the antibody is selected from thegroup consisting of an immunoglobulin, a Bi-specific T-cell engager(BiTE), a diabody, a dual affinity re-targeting (DART), a Fab, a F(ab′),a single chain variable fragment (scFv), and a nanobody.

Embodiment 157. The method of embodiment 153, wherein the transgeneencodes a CAR.

Embodiment 158. The method of embodiment 157, wherein the CAR binds to acancer antigen.

Embodiment 159. The method of any one of embodiments 120-156, whereinthe T cell is sensitized to a target antigen.

Embodiment 160. The method of any one of embodiments 120-159, wherein atransgene (hereinafter “reporter transgene”) encoding a reportermolecule is integrated within the genome of the T cell such thatexpression of the reporter transgene is under control of a promoter,preferably an endogenous promoter of the T cell.

Embodiment 161. The method of any one of embodiments 120-160 which isderived from a human.

Embodiment 162. The method of embodiment 161, wherein the T cell is aprimary human T cell, a T cell derived from a CD34 hematopoietic stemcell, a T cell derived from an embryonic stem cell, or a T cell derivedfrom an induced pluripotent stem cell.

Embodiment 163. The method of any one of embodiments 120-162, whereinthe transgene is integrated into the first site by targeted homologousrecombination.

Embodiment 164. The method of embodiment 163, wherein the targetedhomologous recombination is carried out by a method comprising using azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), a clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, pyrogen, Aureus,Meganuclease or a Mega-Tal.

Embodiment 165. The T cell of any one of embodiments 120-164, whereinthe transgene is integrated at a plurality of sites within the genome ofthe T cell, and such that expression of the transgene at said pluralityof sites is under the control of different endogenous promoters.

Embodiment 166. The T cell of any one of embodiments 120-165, whereinthe T cell is an immunostimulatory T cell.

Embodiment 167. The T cell of embodiment 166, wherein the T cell isselected from the group consisting of cytotoxic T lymphocyte (CTL), CD4+subtype, CD8+ subtype, central memory T cell (TCM), stem memory T cell(TSCM), effector memory T cell, effector T cell, Th1 cell, Th2 cell, Th9cell, Th17 cell, Th22 cell, and Tfh (follicular helper) cell.

Embodiment 168. The T cell of embodiment 167, wherein the T cell isCD4+.

Embodiment 169. The T cell of embodiment 167, wherein the T cell isCD8+.

Embodiment 170. The method of any one of embodiments 120-169, whereinthe subject has cancer.

Embodiment 171. The method of embodiment 170, wherein the cancer isleukemia.

Embodiment 172. The method of any one of embodiments 120-170, whereinthe subject has a tumor.

Embodiment 173. The method of any one of embodiments 120-172, whereinthe subject is a human, and wherein the cell is derived from a human.

Embodiment 174. The method of any one of embodiments 120-173, whereinthe cell is autologous to the subject.

Embodiment 175. The method of any one of embodiments 120-173, whereinthe cell is non-autologous to the subject.

Embodiment 176. A method of treating a subject with T cell therapy inneed thereof, wherein the subject is in need of an inhibited immuneresponse, comprising administering to the subject a therapeuticallyeffective amount of a cell or population of cells, wherein the cell is aT cell wherein a transgene is integrated at a first site within thegenome of the cell such that expression of the transgene is undercontrol of an endogenous promoter of the T cell, wherein the transgeneencodes a therapeutic protein or therapeutic nucleic acid.

Embodiment 177. The method of embodiment 176, wherein the cell or cellpopulation is administered as a pharmaceutical composition.

Embodiment 178. The method of embodiment 176, wherein the transgeneencodes a therapeutic protein.

Embodiment 179. The method of embodiment 176, wherein the transgeneencodes a therapeutic nucleic acid.

Embodiment 180. The method of any one of embodiments 176-179, whereinthe transgene is integrated at a single site within the genome.

Embodiment 181. The method of any one of embodiments 176-179, whereinthe transgene is integrated at two sites within the genome of the cell.

Embodiment 182. The method of any one of embodiments 176-181, whereinthe first site is an an exon of the endogenous gene under control of theendogenous promoter.

Embodiment 183. The method of embodiment 182, wherein the first site iswithin the first exon of the endogenous gene.

Embodiment 184. The method of any one of embodiments 176-183, whereinthe endogenous promoter is constitutive.

Embodiment 185. The method of embodiment 184, wherein the promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

Embodiment 186. The method of any one of embodiments 176-183, whereinthe endogenous promoter is active in a subset of T cells.

Embodiment 187. The method of embodiment 186, wherein the endogenouspromoter is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, CD3z promoter, actin promoter, CD25promoter, IL2 promoter, CD69 promoter, GzmB promoter, T-bet promoter,IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter,IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter,CD95 promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1promoter, CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DRpromoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter,CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter,CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter,Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter,IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

Embodiment 188. The method of any one of embodiments 176-183, whereinthe endogenous promoter is inducible.

Embodiment 189. The method of embodiment 188, wherein the endogenouspromoter is induced by activation of the T cell.

Embodiment 190. The method of embodiment 188, wherein the promoter isinduced by binding of a chimeric antigen receptor (CAR), a chimericco-stimulatory receptor (CCR), T cell receptor (TCR), CD28, CD27, or4-1BB expressed by the T cell to its respective binding partner.

Embodiment 191. The method of embodiment 190, wherein the promoter isinduced by binding of a CAR, CCR or TCR expressed by the T cell to itsrespective binding partner.

Embodiment 192. The method of embodiment 191, wherein the promoter isselected from the group consisting of nuclear factor of activated Tcells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cellimmunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyteantigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3)promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and2B4 promoter.

Embodiment 193. The method of embodiment 188, wherein the promoter isinduced by binding of a ligand to an inhibitory receptor expressed bythe T cell.

Embodiment 194. The method of embodiment 193, wherein the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4.

Embodiment 195. The method of embodiment 193, wherein the promoter isselected from the group consisting of CPT1a promoter and ATGL promoter.

Embodiment 196. The method of embodiment 188, wherein the promoter isinduced by binding of a cytokine to a cytokine receptor expressed by theT cell.

Embodiment 197. The method of embodiment 196, wherein the cytokine isselected from the group consisting of interleukin 10 (IL10) andtransforming growth factor β (TGFβ).

Embodiment 198. The method of embodiment 196, wherein the promoter isselected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

Embodiment 199. The method of embodiment 188, wherein the promoter isinduced by contact of the cell with a nucleic acid.

Embodiment 200. The method of embodiment 199, wherein the nucleic acidis selected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA.

Embodiment 201. The method of embodiment 200, wherein the promoter isselected from the group consisting of Type I interferon (IFN) alpha,Type I IFN beta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1, and IL6.

Embodiment 202. The method of embodiment 188, wherein the promoter isinduced by contact of the cell with a metabolite.

Embodiment 203. The method of embodiment 202, wherein the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

Embodiment 204. The method of embodiment 188, wherein the promoter isinduced by a metabolic change in the cell or contact of the cell with asubstance that causes a metabolic change in the cell.

Embodiment 205. The method of embodiment 204, wherein the promoter isPKM2 promoter.

Embodiment 206. The method of embodiment 188, wherein the promoter isinduced by a particular ion concentration in the cell or contact of thecell with a particular ion concentration.

Embodiment 207. The method of embodiment 206, wherein the ion ispotassium or calcium.

Embodiment 208. The method of embodiment 206, wherein the promoter isselected from the group consisting of IL2 promoter, TNFalpha promoter,and IFNgamma promoter.

Embodiment 209. The method of any one of embodiments 176-208, whereinthe transgene encodes a molecule selected from the group consisting of aCAR, a CCR, a cytokine, a dominant negative, a microenvironmentmodulator, an antibody, a biosensor, a chimeric receptor ligand (CRL), achimeric immune receptor ligand (CIRL), a soluble receptor, a solutetransporter, an enzyme, a ribozyme, a genetic circuit, an epigeneticmodifier, a transcriptional activator, a transcriptional repressor, andnon-coding RNA.

Embodiment 210. The method of embodiment 209, wherein the transgeneencodes a cytokine, and optionally the cytokine is immunoinhibitory.

Embodiment 211. The method of embodiment 210, wherein the cytokine isimmunoinhibitory, and the cytokine is selected from the group consistingof TGFBeta and IL10.

Embodiment 212. The method of embodiment 209, wherein the transgeneencodes an antibody, and optionally the antibody is selected from thegroup consisting of an immunoglobulin, a Bi-specific T-cell engager(BiTE), a diabody, a dual affinity re-targeting (DART), a Fab, a F(ab′),a single chain variable fragment (scFv), and a nanobody.

Embodiment 213. The method of embodiment 209, wherein the transgeneencodes a CAR.

Embodiment 214. The method of embodiment 213, wherein the CAR binds to acancer antigen.

Embodiment 215. The method of any one of embodiments 176-212, whereinthe T cell is sensitized to a target antigen.

Embodiment 216. The method of any one of embodiments 176-215, wherein atransgene (hereinafter “reporter transgene”) encoding a reportermolecule is integrated within the genome of the T cell such thatexpression of the reporter transgene is under control of a promoter,preferably an endogenous promoter of the T cell.

Embodiment 217. The method of any one of embodiments 176-216 which isderived from a human.

Embodiment 218. The method of embodiment 217, wherein the T cell is aprimary human T cell, a T cell derived from a CD34 hematopoietic stemcell, a T cell derived from an embryonic stem cell, or a T cell derivedfrom an induced pluripotent stem cell.

Embodiment 219. The method of any one of embodiments 176-218, whereinthe transgene is integrated into the first site by targeted homologousrecombination.

Embodiment 220. The method of embodiment 219, wherein the targetedhomologous recombination is carried out by a method comprising using azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), a clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, pyrogen, Aureus,Meganuclease or a Mega-Tal.

Embodiment 221. The method of any one of embodiments 176-220, whereinthe transgene is integrated at a plurality of sites within the genome ofthe T cell, and such that expression of the transgene at said pluralityof sites is under the control of different endogenous promoters.

Embodiment 222. The method of any one of embodiments 176-221, whereinthe T cell is an immunoinhibitory T cell.

Embodiment 223. The method of embodiment 222, wherein the T cell is aregulatory T cell.

Embodiment 224. The method of any one of embodiments 176-223, whereinthe subject is a human, and wherein the cell is derived from a human.

Embodiment 225. The method of any one of embodiments 176-224, whereinthe cell is autologous to the subject.

Embodiment 226. The method of any one of embodiments 176-224, whereinthe cell is non-autologous to the subject.

Embodiment 227. A method of generating a T cell that expresses atherapeutic transgene, comprising:

introducing into a T cell:

(i) a transgene, and

(ii) a homologous recombination system suitable for targeted integrationof the transgene at a site within the genome of the cell, whereby thehomologous recombination system integrates the transgene at said sitewithin the genome of the cell, and wherein expression of the transgeneis under the control of an endogenous promoter, wherein the transgeneencodes a therapeutic protein or a therapeutic nucleic acid.

Embodiment 228. The method of embodiment 227, wherein the transgeneencodes a therapeutic protein.

Embodiment 229. The method of embodiment 227, wherein the transgeneencodes a therapeutic nucleic acid.

Embodiment 230. The method of embodiment 227, wherein the endogenouspromoter is constitutive.

Embodiment 231. The method of embodiment 230, wherein the promoter isselected from the group consisting of CD4 promoter, CD8a promoter, CD8bpromoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter,CD3e promoter, and CD3z promoter.

Embodiment 232. The method of embodiment 227, wherein the endogenouspromoter is active in a subset of T cells.

Embodiment 233. The method of embodiment 232, wherein the endogenouspromoter is selected from the group consisting of CD4 promoter, CD8apromoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter,CD3g promoter, CD3e promoter, CD3z promoter, actin promoter, CD25promoter, IL2 promoter, CD69 promoter, GzmB promoter, T-bet promoter,IFNgamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL5promoter, IL13 promoter, IL10 promoter, IL17A promoter, IL6 promoter,IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter,CD95 promoter, CD28 promoter, CD27 promoter, CD127 promoter, PD-1promoter, CD122 promoter, CD132 promoter, KLRG-1 promoter, HLA-DRpromoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter,CD58 promoter, CD99 promoter, CD62L promoter, CD103 promoter, CCR4promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter,CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter,Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter,IL-18Ra promoter, c-Kit promoter, and CD130 promoter.

Embodiment 234. The method of embodiment 227, wherein the endogenouspromoter is inducible.

Embodiment 235. The method of embodiment 234, wherein the endogenouspromoter is induced by activation of the T cell.

Embodiment 236. The method of embodiment 234, wherein the promoter isinduced by binding of a chimeric antigen receptor (CAR), a chimericco-stimulatory receptor (CCR), T cell receptor (TCR), CD28, CD27, and4-1BB expressed by the T cell to its respective binding partner.

Embodiment 237. The method of embodiment 236, wherein the promoter isinduced by binding of a CAR, CCR or TCR expressed by the T cell to itsrespective binding partner.

Embodiment 238. The method of embodiment 237, wherein the promoter isselected from the group consisting of nuclear factor of activated Tcells (NFAT) promoter, programmed death 1 (PD-1) promoter, T cellimmunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyteantigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3)promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, and2B4 promoter.

Embodiment 239. The method of embodiment 234, wherein the promoter isinduced by binding of a ligand to an inhibitory receptor expressed bythe T cell.

Embodiment 240. The method of embodiment 239, wherein the inhibitoryreceptor is selected from the group consisting of PD-1, CTLA4, TRAIL,LAG-3, BTLA, TIM-3, Fas, TIGIT, and 2B4.

Embodiment 241. The method of embodiment 239, wherein the promoter isselected from the group consisting of CPT1a promoter and ATGL promoter.

Embodiment 242. The method of embodiment 234, wherein the promoter isinduced by binding of a cytokine to a cytokine receptor expressed by theT cell.

Embodiment 243. The method of embodiment 242, wherein the cytokine isselected from the group consisting of interleukin 2 (IL2), interleukin 7(IL7), interleukin 15 (IL15), and interleukin 21 (IL21).

Embodiment 244. The method of embodiment 242, wherein the cytokine isselected from the group consisting of interleukin 10 (IL10) andtransforming growth factor 13 (TGFI3).

Embodiment 245. The method of embodiment 242, wherein the promoter isselected from the group consisting of T-bet promoter, Eomes promoter,GATA3 promoter, and CD45RA promoter.

Embodiment 246. The method of embodiment 234, wherein the promoter isinduced by contact of the cell with a nucleic acid.

Embodiment 247. The method of embodiment 246, wherein the nucleic acidis selected from the group consisting of viral DNA, viral, RNA, andintracellular microRNA.

Embodiment 248. The method of embodiment 247, wherein the promoter isselected from the group consisting of Type I interferon (IFN) alpha,Type I IFN beta, IRF3, IRF7, NFkB, AP-1, TNF-alpha, IL1, and IL6.

Embodiment 249. The method of embodiment 234, wherein the promoter isinduced by contact of the cell with a metabolite.

Embodiment 250. The method of embodiment 249, wherein the metabolite isselected from the group consisting of pyruvate, glutamine, andbeta-hydroxybutyrate.

Embodiment 251. The method of embodiment 234, wherein the promoter isinduced by a metabolic change in the cell or contact of the cell with asubstance that causes a metabolic change in the cell.

Embodiment 252. The method of embodiment 251, wherein the promoter isPKM2 promoter.

Embodiment 253. The method of embodiment 234, wherein the promoter isinduced by a particular ion concentration in the cell or contact of thecell with a particular ion concentration.

Embodiment 254. The method of embodiment 253, wherein the ion ispotassium or calcium.

Embodiment 255. The method of embodiment 253, wherein the promoter isselected from the group consisting of IL2 promoter, TNFalpha promoter,and IFNgamma promoter.

Embodiment 256. The method of any one of embodiments 227-255, whereinthe transgene encodes a molecule selected from the group consisting of aCAR, a CCR, a cytokine, a dominant negative, a microenvironmentmodulator, an antibody, a biosensor, a chimeric receptor ligand (CRL), achimeric immune receptor ligand (CIRL), a soluble receptor, a solutetransporter, an enzyme, a ribozyme, a genetic circuit, an epigeneticmodifier, a transcriptional activator, a transcriptional repressor, andnon-coding RNA.

Embodiment 257. The method of embodiment 256, wherein the transgeneencodes a cytokine, and optionally the cytokine is immunostimulatory.

Embodiment 258. The method of embodiment 257, wherein the cytokine isimmunostimulatory, and the cytokine is selected from the groupconsisting of IL2, IL12, IL15, and IL18.

Embodiment 259. The method of embodiment 256, wherein the transgeneencodes a cytokine, and optionally the cytokine is immunoinhibitory.

Embodiment 260. The method of embodiment 259, wherein the cytokine isimmunoinhibitory, and the cytokine is selected from the group consistingof TGFBeta and IL10.

Embodiment 261. The method of embodiment 256, wherein the transgeneencodes an antibody, and optionally the antibody is selected from thegroup consisting of an immunoglobulin, a Bi-specific T-cell engager(BiTE), a diabody, a dual affinity re-targeting (DART), a Fab, a F(ab′),a single chain variable fragment (scFv), and a nanobody.

Embodiment 262. The method of embodiment 256, wherein the transgeneencodes a CAR.

Embodiment 263. The method of embodiment 262, wherein the CAR binds to acancer antigen.

Embodiment 264. The method of any one of embodiments 227-263, whereinthe T cell is sensitized to a target antigen.

Embodiment 265. The method of any one of embodiments 227-264, wherein atransgene (hereinafter “reporter transgene”) encoding a reportermolecule is integrated within the genome of the T cell such thatexpression of the reporter transgene is under control of a promoter,preferably an endogenous promoter of the T cell.

Embodiment 266. The method of any one of embodiments 227-265 which isderived from a human.

Embodiment 267. The method of embodiment 266, wherein the T cell is aprimary human T cell, a T cell derived from a CD34 hematopoietic stemcell, a T cell derived from an embryonic stem cell, or a T cell derivedfrom an induced pluripotent stem cell.

Embodiment 268. The method of any one of embodiments 227-267, whereinthe transgene is integrated into the first site by targeted homologousrecombination.

Embodiment 269. The method of embodiment 268, wherein the targetedhomologous recombination is carried out by a method comprising using azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), a clustered regularly-interspersed short palindromicrepeats (CRISPR) associated protein 9 (Cas9), Cpf1, pyrogen, Aureus,Meganuclease or a Mega-Tal.

Embodiment 270. The method of any one of embodiments 227-269, whereinthe transgene is integrated at a plurality of sites within the genome ofthe T cell, and such that expression of the transgene at said pluralityof sites is under the control of different endogenous promoters.

Embodiment 271. The method of any one of embodiments 227-270, whereinthe transgene that is introduced into the cell is contained in atargeting construct.

Embodiment 272. The method of embodiment 271, wherein the targetingconstruct comprises viral nucleic acid sequences.

Embodiment 273. The method of embodiment 271 or 272, wherein thetargeting construct is packaged into a natural or recombinantadeno-associated virus (AVV) viral particle.

Embodiment 274. The method of embodiment 273, wherein the AAV particlecomprises AAV6 sequences.

Embodiment 275. The method of embodiment 271 or 272, wherein thetargeting construct is packaged into a non-integrating gamma-retrovirus.

Embodiment 276. The method of any one of embodiments 227-275, whereinthe transgene in the targeting construct are not operably linked to apromoter.

Embodiment 277. The method of any one of embodiments 227-276, furthercomprising introducing a second transgene into the T cell.

Embodiment 278. The method of embodiment 277, wherein the firsttransgene is under control of an endogenous constitutive promoter andthe second transgene is under control of an endogenous induciblepromoter.

Embodiment 279. The method of embodiment 278, wherein the firsttransgene is a CAR.

Embodiment 280. The method of embodiment 279, wherein the endogenousconstitutive promoter is a T cell receptor promoter.

Embodiment 281. The method of embodiment 280, wherein the promoter isselected from the group consisting of T cell receptor alpha chainpromoter, T cell receptor beta chain promoter, CD3 gamma chain promoter,CD3 delta chain promoter, CD3 epsilon chain promoter, and CD3 zeta chainpromoter.

Embodiment 282. The method of embodiment 281, wherein the promoter is Tcell receptor alpha chain promoter.

Embodiment 283. A vector comprising a non-integrating gamma-retrovirus.

Embodiment 284. The vector of embodiment 283, wherein thenon-integrating gamma-retrovirus comprises a mutated integrase.

Embodiment 285. The vector of embodiment 284, wherein the mutatedintegrase is mutated at a DDE motif.

Embodiment 286. The vector of embodiment 285, wherein the mutatedintegrase has a mutation selected from the group consisting of D124A,D124E, D124N, D124V, D183A, D183N, D124A and D183A, D124A and D183N,D124E and D183A, D124E and D183N, D124N and D183A, D124N and D183N,D124V and D183A, and D124V and D183N.

Embodiment 287. A T cell wherein a recombinant nucleic acid sequenceencoding a chimeric antigen receptor (CAR) is integrated at a first sitewithin the genome of the cell such that the CAR is expressed by the cellat the surface of the cell, and wherein integration of the nucleic acidencoding the CAR at said first site reduces or prevents expression of afunctional T cell receptor (TCR) complex at the surface of the cell.

Embodiment 288. The T cell of embodiment 287, wherein the nucleic acidsequence encoding the CAR is integrated at a single site within thegenome.

Embodiment 289. The T cell of embodiment 287, wherein the nucleic acidsequence encoding the CAR is integrated at two sites within the genomeof the cell.

Embodiment 290. The T cell of embodiment 289, wherein the first site isan an exon of the gene encoding a protein of the TCR complex.

Embodiment 291. The T cell of any one of embodiments 287-290, whereinintegration of the nucleic acid sequence encoding the CAR at the firstsite reduces or prevents expression of a protein selected from the groupconsisting of T cell receptor alpha chain, T cell receptor beta chain,CD3 gamma chain, CD3 delta chain, CD3 epsilon chain, and CD3 zeta chain.

Embodiment 292. The T cell of any one of embodiments 287-291, whereinexpression of the integrated nucleic acid sequence in the T cell isunder the control of an endogenous promoter.

Embodiment 293. The T cell of embodiment 292, wherein the endogenouspromoter is a T cell receptor complex promoter.

Embodiment 294. The T cell of embodiment 292, wherein the endogenouspromoter is a promoter of a gene encoding a T cell receptor alpha chain,T cell receptor beta chain, CD3 gamma chain, CD3 delta chain, CD3epsilon chain, or CD3 zeta chain.

Embodiment 295. The T cell of any one of embodiments 287-294, whereinthe CAR binds to a cancer antigen.

Embodiment 296. The T cell of any one of embodiments 287-295, whereinthe T cell is selected from the group consisting of cytotoxic Tlymphocyte (CTL), CD4+ subtype, CD8+ subtype, central memory T cell(TCM), stem memory T cell (TSCM), effector memory T cell, effector Tcell, Th1 cell, Th2 cell, Th9 cell, Th17 cell, Th22 cell, Tfh(follicular helper) cell, and T regulatory cell.

Embodiment 297. The T cell of any one of embodiments 287-296 which isderived from a human.

Embodiment 298. The T cell of embodiment 297, wherein the T cell is aprimary human T cell, a T cell derived from a CD34 hematopoietic stemcell, a T cell derived from an embryonic stem cell, or a T cell derivedfrom an induced pluripotent stem cell.

Embodiment 299. The T cell of any one of embodiments 287-298, whereinthe nucleic acid sequence encoding the CAR is integrated into the firstsite by targeted homologous recombination.

Embodiment 300. The T cell of embodiment 299, wherein the targetedhomologous recombination is carried out using a zinc-finger nuclease(ZFN), a transcription activator-like effector nuclease (TALEN),aclustered regularly-interspersed short palindromic repeats (CRISPR)associated protein 9 (Cas9), Cpf1, Meganuclease or a Mega-Tal.

Embodiment 301. The T cell of embodiment 287, wherein the nucleic acidsequence encoding the CAR is integrated at a plurality of sites withinthe genome of the cell, and such that expression of the nucleic acidsequence encoding the CAR at said plurality of sites is under thecontrol of a different endogenous promoter.

Embodiment 302. The T cell of any one of embodiments 287-301, whereinsaid nucleic acid sequence encoding a CAR is also integrated at a secondsite within the genome of the cell such that the CAR is expressed by thecell at the surface of the cell.

Embodiment 303. The T cell of embodiment 302, wherein integration of thenucleic acid encoding the CAR at said second site also reduces orprevents expression of a functional TCR complex at the surface of thecell, wherein said first site and said second site are in differentgenes.

Embodiment 304. The T cell of any one of embodiments 287-303, wherein asecond nucleic acid sequence encoding a second CAR is integrated at asecond site within the genome of the cell such that the second CAR isexpressed by the cell at the surface of the cell, and such thatexpression of the second nucleic acid sequence is under the control ofan endogenous promoter at said second site, wherein said first site andsaid second site are in different genes.

Embodiment 305. A human T cell wherein a promotor-less recombinantnucleic acid sequence encoding a CAR is integrated at a site in thegenome of the cell, said site being the first exon of the TCR alphachain, such that the CAR is expressed under control of the endogenousTCR alpha chain promoter, to produce said CAR at the surface of thecell, and wherein integration of the CAR at said site reduces orprevents expression of a functional TCR alpha chain.

Embodiment 306. The human T cell of embodiment 305, wherein the CARbinds to CD19.

Embodiment 307. An isolated population of T cells, which comprises aplurality of the cell of any one of embodiments 287-306.

Embodiment 308. A pharmaceutical composition comprising atherapeutically effective amount of the cell of any one of embodiments287-306; and a pharmaceutically acceptable carrier.

Embodiment 309. A pharmaceutical composition comprising atherapeutically effective amount of a population of T cells, whichpopulation comprises a plurality of the cell of any one of embodiments287-306; and a pharmaceutically acceptable carrier.

Embodiment 310. A method of treating a subject with CAR therapy in needthereof, comprising administering to the subject a therapeuticallyeffective amount of the cell of any one of embodiments 287-306.

Embodiment 311. A method of treating a subject with CAR therapy in needthereof, comprising administering to the subject the pharmaceuticalcomposition of embodiment 308.

Embodiment 312. A method of treating a subject with CAR therapy in needthereof, comprising administering to the subject a therapeuticallyeffective amount of the cell population of embodiment 307.

Embodiment 313. A method of treating a subject with CAR therapy in needthereof, comprising administering to the subject the pharmaceuticalcomposition of embodiment 309.

Embodiment 314. The method of any one of embodiments 310-313, whereinthe subject has cancer, and wherein the CAR binds to a cancer antigen ofthe cancer.

Embodiment 315. The method of embodiment 314, wherein the cancer isleukemia.

Embodiment 316. The method of any one of embodiments 310-314, whereinthe subject has a tumor.

Embodiment 317. The method of any one of embodiments 310-316, whereinthe subject is a human, and wherein the cell is derived from a human.

Embodiment 318. The method of any one of embodiments 310-317, whereinthe cell is autologous to the subject.

Embodiment 319. The method of any one of embodiments 310-317, whereinthe cell is non-autologous to the subject.

Embodiment 320. A method of generating a T cell that expresses achimeric antigen receptor (CAR) and lacks a functional T cell receptor(TCR) complex, comprising:

introducing into a T cell:

(i) a nucleic acid sequence encoding a CAR, and

(ii) a homologous recombination system suitable for targeted integrationof the nucleic acid sequence at a site within the genome of the cell,whereby the homologous recombination system integrates the nucleic acidsequence encoding the CAR at said site within the genome of the cellsuch that integration of the CAR at said site reduces or preventsexpression of a functional T cell receptor complex at the surface of thecell, thereby generating a T cell that expresses the CAR and lacks afunctional TCR complex.

Embodiment 321. The method of embodiment 320, wherein expression of theCAR is under the control of an endogenous promoter.

Embodiment 322. The method of embodiment 321, wherein the endogenouspromoter is a promoter of a gene encoding a T cell receptor alpha chain,T cell receptor beta chain, CD3 gamma chain, CD3 delta chain, CD3epsilon chain, or CD3 zeta chain.

Embodiment 323. The method of any one of embodiments 320-322, whereinthe homologous recombination system comprises a zinc-finger nuclease(ZFN), a transcription activator-like effector nuclease (TALEN), orclustered regularly-interspersed short palindromic repeats (CRISPR)associated protein 9 (Cas9), Cpf1, Meganuclease or a Mega-Tal.

Embodiment 324. The method of any one of embodiments 320-323, whereinthe nucleic acid sequence encoding the CAR that is introduced into thecell is contained in a targeting construct.

Embodiment 325. The method of embodiment 324, wherein the targetingconstruct comprises adeno-associated virus 2 (AAV2) sequences.

Embodiment 326. The method of embodiment 324 or 325, wherein thetargeting construct is packaged into a natural or recombinantadeno-associated virus (AVV) viral particle.

Embodiment 327. The method of embodiment 326, wherein the AAV particlecomprises AAV6 sequences.

Embodiment 328. The method of any one of embodiments 320-327, whereinthe nucleic acid sequences encoding the CAR in the targeting constructare not operably linked to a promoter.

Embodiment 329. The method of any one of embodiments 320-328, whereinthe targeting construct comprises in 5′ to 3′ order: a first viralsequence, a left homology arm, a nucleic acid sequence encoding aself-cleaving porcine teschovirus 2A, the nucleic acid sequence encodingthe CAR, a polyadenylation sequence, a right homology arm, and a secondviral sequence.

Embodiment 330. The method of embodiment 329, wherein the first or thesecond viral sequence is from an adeno-associated virus (AAV).

Embodiment 331. The method of embodiment 330, wherein the AAV is AAV2,AAVS or AAV6.

Embodiment 332. An induced pluripotent stem cell, wherein a recombinantnucleic acid sequence encoding a chimeric antigen receptor (CAR) isintegrated at a first site within the genome of the cell such that theCAR is expressed by the cell at the surface of the cell, and whereinintegration of the nucleic acid encoding the CAR at said first sitereduces or prevents expression of a functional T cell receptor (TCR)complex at the surface of the cell.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

8. EXAMPLES 8.1 Example 1 One-Step Genearation of Universal CAR T Cells

Described below is a strategy for one-step generation of universal CARTcells.

The method involves a two-in-one genome editing strategy to generateuniversal CAR T cells with the CAR under the control of the TCR alphapromoter. To do so, the TCR expression was disrupted by targeting a CARinto the TCR alpha constant chain gene and the endogenous promoter wasused to express the CAR. Tailored nucleases (TALEN and CRISPR/cas9) weredesigned targeting the first exon of the TRAC gene, and an AAV vectorwas used to promote integration of the CAR in frame with the TRAC geneby homologous directed repair (HDR).

Tailored Nucleases: TALEN and a gRNA were designed to target the firstexon on the TRAC gene. The sequence targeted is located upstream of thetransmembrane domain of the TCR alpha. This domain is required for theTCR alpha and beta assembly and addressing to the cell surface.Non-homologous end joining (NHEJ) or integration of the CAR by HDR atthis locus efficiently disrupts the TCR complex.

TRAC-gRNA sequence: (SEQ ID NO: 24)C*A*G*GGUUCUG GAUAUCUGUG UUUUAGAGCU AGAAAUAGCAAGUUAAAAUA AGGCUAGUCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCU*U*U*U*2′-O-methyl 3′ phosphorothioateTALEN target sequence (spacer underlined): (SEQ ID NO: 25)TTGTCCCACAGATATCC AGAACCCTGACCCTG CCGTGTACCAGCTGAGA

Messenger RNA: The plasmids coding for the TALEN were synthesized byTransposagen and linearized with AgeI. TALEN mRNA were transcribed andpolyadenylated in vitro using the mMessage mMachine T7 Ultra kit (LifeTechnologies; Carlsbad, Calif.). RNA was purified with RNeasy columns(Qiagen; Valencia, Calif.) and quantified using the Nanodrop machine.Quality of the RNA was verified on a denaturing formaldehyde/MOPSagarose gel. Modified guide RNAs (gRNAs) and Cas9 mRNA were synthesizedby TriLink Biotechnologies. gRNAs were reconstituted at 1 ug/uL incytoporation T Buffer (Harvard Apparatus; Holliston, Mass.).

AAV: For TRAC targeting (see FIG. 1A), based on a pAAV-GFP backbone(Cellbiolabs; San Diego, Calif.), the pAAV-TRAC-P2A-1928z was designedand cloned containing 1.9 kb of genomic TRAC (amplified by PCR) flankingthe TALEN and gRNA targeting sequences, a self-cleaving P2A peptide inframe with the first exon of TRAC, followed by the 1928z CAR used inclinical trials (Brentjens et al., Sci. Transl. Med. 5(177):177ra38.doi: 10.1126/scitranslmed.3005930 (2013)). Briefly, the CAR comprises asingle chain variable fragment 19 scFV, specific for the human CD19preceded by a CD8a leader peptide and followed by CD28hinge-transmembrane-intracellular regions and CD3 intracellular domain.The cassette is terminated by the bovine growth hormone polyA signal(BGHpA).

Cells: Buffy coats from healthy volunteer donors were obtained from theNew York Blood Center. Peripheral blood mononuclear cells were isolatedby density gradient centrifugation, and T lymphocytes were then purifiedusing the Pan T cells isolation kit (Miltenyi Biotech; San Diego,Calif.). Cells were activated with Dynabeads (1:1 beads:Cell) HumanT-Activator CD3/CD28 (ThermoFisher; Carlsbad Calif.) in X-vivo 15 medium(Lonza; Basel, Switzerland) supplemented human serum (GeminiBioproducts; West Sacramento, Calif.) with 200 U/ml IL-2 (MiltenyiBiotech) at a density of 10⁶ cells/ml. The medium was changed every 2days, and cells were replated at 10⁶ cells/ml.

Gene Targeting: After a 48 h-activation, the CD3/CD28 beads weremagnetically removed, and the cells were cultured in the absence ofbeads for 12-16 hours. T lymphocytes were transfected by electrotransferof TALEN or Cas9/gRNA RNAs using an AgilePulse MAX system (HarvardApparatus). Briefly, cells were washed in cytoporation medium T (HarvardApparatus). Cells were then pelleted, resuspended in cytoporation mediumT at 30×10⁶ cells/ml. 3×10⁶ cells were mixed with the indicated dose ofeach mRNA encoding the tailored nucleases into a 0.2 cm cuvette. Theelectroporation consisted of two 0.1 ms pulses at 600 V followed by four0.2ms pulses at 100V. Following electroporation, cells were diluted intoculture medium and incubated at 37° C., 5% CO₂. AAV was added to theculture 2 to 4 hours after electroporation, followed by continued 30° C.incubation for 20 additional hours. AAV donor was added at the indicatedMOI (1e5 to 1e6 MOI). Subsequently, edited cells were cultured usingstandard conditions (37° C. and expanded in T cell growth medium,replenished as needed to maintain a density of ˜1e6 cells/ml every 2 to3 days).

These conditions are highly reproducible among donors and resulted in upto 50% of TCR−/CAR+ T cells in one single step with both TALEN andCRISPR.

To obtain TCR-negative T cells, TCR-positive T cells were removed fromthe culture using magnetic PE-anti-TCRab and anti-PE microbeads and LScolumns (Miltenyi Biotech).

Retroviral Vector Constructs and Retroviral Production: Plasmidsencoding the SFG γ-retroviral vector (Rivière et al., Proc. Natl. Acad.Sci. USA 92(15):6733-6737 (1995)) were prepared using standard molecularbiology techniques. Synthesis of SFG-1928z and SFG-P28z has beenpreviously described (Brentjens et al., Nat Med. 9(3):279-286 (2003),Brentjens et al., 2007 Clin. Cancer Res. 13(18 Pt 1):5426-5435 (2007);Maher et al., Nat. Biotechnol. 20(1):70-5 (2002)). VSV-G pseudotypedretroviral supernatants derived from transduced gpg29 fibroblasts (H29)were used to construct stable retroviral-producing cell lines aspreviously described (Gong et al., Neoplasia 1:123-127 (1999)).

Retroviral Transduction: T cells were transduced on two consecutive daysby centrifugation on Retronectin (Takara; Mountain View, Calif.)-coatedoncoretroviral vector-bound plates.

Cytotoxicity assays: The cytotoxicity of T cells transduced with a CARwas determined by standard luciferase-based assay. In brief, NALM6expressing firefly luciferase-GFP served as target cells. The effector(E) and tumor target (T) cells were co-cultured in triplicates atindicated E/T ratio using black-walled 96 well plates with 1×10⁵ targetcells in a total volume of 100 μl/well in NALM6 Medium. Target cellsalone were plated at the same cell density to determine the maximalluciferase expression (relative light units; RLUmax). 18 hr later, 100μl luciferase substrate (Bright-Glo, Promega; Madison, Wis.) wasdirectly added to each well. Emitted light was detected in aluminescence plate reader or Xenogen IVIS Imaging System (Xenogen;Alameda, Calif.), and quantified using Living Image software (Xenogen).Lysis was determined as [1−(RLUsample)/(RLUmax)]×100.

Mouse Systemic Tumor Model: The mouse model used was 8- to 12-week-oldNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory; BarHarbor, Me.), under a protocol approved by the MSKCC InstitutionalAnimal Care and Use Committee. Mice were inoculated with 0.5×10⁶FFLuc-GFP NALM6 cells by tail vein injection, followed by 2×10⁵ CAR Tcells injected four days later. NALM6 produce very even tumor burdensand no mice were excluded prior to treatment. No blinding method wasused. Bioluminescence imaging utilized the Xenogen IVIS Imaging System(Xenogen) with Living Image software (Xenogen) for acquisition ofimaging datasets. Tumor burden was assessed as previously described(Gade et al., Cancer Res. 65(19):9080-9088 (2005)).

FIG. 1A shows a schematic of tailored nuclease (TALEN orCRISPR/Cas9)-induced targeted integration into TCR alpha constant (TRAC)locus. The targeting construct (AAV6) contains the CAR gene flanked byhomology sequences (left homologous arm, LHA and right homologous arm,RHA). Once integrated CAR expression is driven by the endogenous TCRapromoter while TRAC locus is disrupted (TRAV: TCR alpha variable region;TRAJ: TCR alpha joining region; 2A: the self cleaving Porcineteschovirus 2A sequence; pA: bovine growth hormone PolyA sequence). FIG.1B shows representative TCR/CAR flow plot 5 days after transfection of Tcells with TRAC TALEN mRNA and addition of AAV6 at the noted MOI. Asshown in FIG. 1B, the expression of CAR increased and the expression ofTCR decreased with increasing AAV MOI. FIG. 1C shows a bar-graph of thepercentage of TCR disruption (KO: knockout) and targeted integration(KI: knockin) depending on the AAV6 MOI. The percentages were assessedby FACS analysis. FIG. 1D shows average CAR expression mean fluorescenceintensity (MFI) 5 days after CAR vectorization into T cells (n=6 to 8independent experiments). The results show that targeted integration ofCAR in the TRAC locus resulted in a homogenous population of T cellswith similar expression levels of CAR. FIG. 1E shows coefficient ofvariation of the CAR+ T cells measuring the dispersion in the CARexpression (ratio of the standard deviation to the mean).TRAC-P2A-1928z: Targeted integration into

TRAC. SFG-1928z: semi-random integration using the SFG retrovirus.****P<0.0001 (unpaired T-test). These results show that targetedintegration of CAR in the TRAC locus resulted in a homogeneouspopulation of T cells with similar expression.

FIG. 2A shows flow cytometry analysis showing CAR and TCR expression.TRAC-P2A-1928z were generated as in FIG. 1. TALEN-generated TCR− cellswere transduced with SFG-1928z retrovirus. TCR+ cells were transducedwith either SFG-1928z or SGF-P28z retrovirus. FIG. 2B shows cumulativecell counts of indicated CAR T cells upon weekly stimulation with CD19+target cells, showing that cells expressing 1928z exhibited in vitroproliferation. FIG. 2C shows cytotoxic activity using an 18 hrbioluminescence assay, using firefly luciferase (FFL)-expressing NALM6as target cells. Cells expressing 1928z exhibited cytotoxic activity.FIGS. 2D and 2E show FFL-NALM6 bearing mice were treated with 2×10⁵ CART cells. Tumor burden is shown as bioluminescent signal quantified peranimal every week over a 40-day period. Quantification is the averagephoton count of ventral and dorsal acquisitions per animal at all giventime points, and it is expressed as radiance. Each line in FIG. 2Erepresents one mouse. n=7 mice per group. The lower right figure isKaplan-Meier analysis of survival of mice in FIGS. 2D and 2E. Theseresults demonstrate that targeted integration of a CAR into TRACresulted in survival significantly longer than with semi-randomintegration using the SFG retrovirus.

Taken together these results show that, at an equivalent dose of CAR Tcell injected, cells with the CAR targeted into the TRAC locus arestrongly more potent than the cells retrovirally transduced with CAR.

As described above, a strategy for one-step generation of universal CART cells was developed by targeting the integration of a promoter-lessCAR gene cassette in the TCR alpha constant chain (TRAC) first exon.This results in CAR expression under the control of the endogenous TCRalpha promoter with concomitant disruption of the TCR alpha geneexpression. As all components of the TCR complex are required for itslocalization to the cytoplasmic membrane, the TCR alpha disruption leadsto TCR negative cells. This approach is suitable with the commonly usedgenome editing platforms (for example, TALEN, CRISPR/Cas9, ZFN) andresults in homologous recombination at the TRAC target site using an AAVdonor template in T cells. The efficiency of TALEN and CRISPR/Cas9 topromote homologous recombination using AAV6 donor template in T cellswas compared. Conditions were established yielding up to 50% ofuniversal CAR T cells combining target gene disruption and CAR targetedinsertion in a single step. The targeted integration of the CARtransgene was molecularly confirmed, which results in highly homogeneousand stable CAR expression in human peripheral blood T cells. These Tcells exhibited the same in vitro tumor lysis activity and proliferationthan retrovirally transduced CAR T cells, which supports theirusefulness in in vivo anti-tumor activity. The endogenous TCR alphapromoter provided unanticipated benefits. The method provided highlyhomogeneous and stable CAR expression in human peripheral blood T cells,and also improved T cell persistence. Most importantly, these T cellsexhibited higher in vitro and in vivo tumor lysis activity,proliferation and persistence than retrovirally transduced CAR T cells,while their Graft versus host disease potential was removed by reducingor preventing expression of a functional T cell receptor complex at thesurface of the cell. The process described herein, which combines thescalability of universal T cell manufacturing with the uniformity andsafety of targeted CAR gene integration, is useful for the developmentof off-the-shelf CAR therapy that can be scaled up and readily providedto patients, as needed.

8.2 Example 2 Targeting a CAR to the TRAC Locus with CRISPR/Cas9Enhances Tumor Rejection

This example shows expression by a T cell of a CAR encoded by atransgene was carried out, wherein the expression of the transgene wasunder the control of an endogenous T cell promoter, specifically thehuman T cell receptor a chain (TRAC) promoter. Described below areexperiments showing that directing a CD19-specific CAR to the human Tcell receptor a chain (TRAC) locus not only result in uniform CARexpression in human peripheral blood T cells, but also enhances T cellpotency, with edited cells vastly outperforming conventionally generatedCAR T cells in a mouse model of acute lymphoblastic leukaemia. It isfurther demonstrated that targeting the CAR to the TRAC locus avertstonic CAR signalling and establishes effective internalization andre-expression of the CAR following single or repeated exposure toantigen, delaying effector T cell differentiation and exhaustion. Thesefindings uncover facets of CAR immunobiology and underscore the vastpotential of CRISPR/Cas9 genome editing to advance immunotherapies.

Methods. Guide-RNA: A guide RNA (gRNA) gRNA was designed to target thefirst exon of the constant chain of the TCRα gene (TRAC). The sequencetargeted is located upstream of the transmembrane domain of the TCRalpha. This domain is required for the TCR alpha and beta assembly andaddressing to the cell-surface. Both non-homologous end joining (NHEJ)and integration of the CAR by HDR at this locus would then efficientlydisrupt the TCR complex.

For the B2M, both a gRNA and a TALEN (Transcription activator-likeeffector nucleases) targeting the first exon of B2M gene were designed,and a higher cutting efficiency was obtained with the TALEN. The sameprotocol was used, similar cytotoxicity and specificity was obtained forboth methods, and the CAR T cells obtained were not discernable in termof activity and proliferation. For manufacturing reasons the B2M TALENwas mainly used in this study.

TRAC-gRNA sequence: (SEQ ID NO: 26)C*A*G*GGUUCUG GAUAUCUGUG UUUUAGAGCU AGAAAUAGCAAGUUAAAAUA AGGCUAGUCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCU*U*U*UB2M-gRNA sequence: (SEQ ID NO: 27)G*G*C*CACGGAG CGAGACAUCU UUUUAGAGCU AGAAAUAGCAAGUUAAAAUA AGGCUAGUCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCU*U*U*U*2′-O-methyl 3′ phosphorothioate B2M-TALEN targeting sequence:(SEQ ID NO: 28) TTAGCTGTGCTCGCGC (TACTCTCTCTTTCTG) GCCTGGAGGCTATCCA.Left TAL effector (spacer) Right TAL effector.

Messenger RNA: Modified guide RNAs (gRNAs) and Cas9 mRNA weresynthesized by TriLink Biotechnologies (San Diego, Calif.). Guide RNAswere reconstituted at 1 μg/μL in cytoporation T Buffer (HarvardApparatus; Holliston, Mass.).

AAV: Based on a pAAV-GFP backbone (Cell Biolabs; San Diego, Calif.), thepAAV-TRAC-1928z was designed and cloned containing 1.9 kb of genomicTRAC (amplified by PCR) flanking the gRNA targeting sequences, aself-cleaving P2A peptide in frame with the first exon of TRAC followedby the 1928z CAR used in clinical trials (Brentj ens et al., Sci. Trans.Med. 5:177ra138 (2013)). Briefly, the CAR comprises a single chainvariable fragment 19 scFV specific for the human CD19 preceded by a CD8aleader peptide and followed by CD28 hinge-transmembrane-intracellularregions and CD3 intracellular domain. The CAR cDNA is followed by thebovine growth hormone polyA signal (bGHpA). When targeting the B2Mlocus, a similar strategy was followed, except that no P2A sequence wasrequired since the 1928z-pA sequence was placed in frame at the ATG ofthe B2M gene. When using exogenous promoters (EF1α, LTR, PGK, orPGK100), the promoter-1928z-pA cassette was placed in reverseorientation at the same TRAC or B2M entry points.

Cells: Buffy coats from healthy volunteer donors were obtained from theNew York Blood Center. Peripheral blood mononuclear cells were isolatedby density gradient centrifugation, and T lymphocytes were then purifiedusing the Pan T cell isolation kit (Miltenyi Biotech; San Diego,Calif.). Cells were activated with Dynabeads (1:1 beads:cell) HumanT-Activator CD3/CD28 (ThermoFisher; Carlsbad, CA) in X-vivo 15 medium(Lonza; Basel, Switzerland) supplemented with 5% human serum (GeminiBioproducts; West Sacramento, Calif.) with 200 U/ml IL-2 (MiltenyiBiotech) at a density of 10⁶ cells/ml. The medium was changed every 2days, and cells were replated at 10⁶ cells/ml.

Gene Targeting: 48 hours after initiating T cell activation, theCD3/CD28 beads were magnetically removed, and the T cells weretransfected by electrotransfer of Cas9 mRNA and gRNA using an AgilePulseMAX system (Harvard Apparatus). 3×10⁶ cells were mixed with 5 μg of Cas9and 5 μg of gRNA into a 0.2 cm cuvette. Following electroporation, cellswere diluted into culture medium and incubated at 37° C./5% CO₂.Recombinant AAV6 donor vector (manufactured by SignaGen; Gaithersburg,Md.) was added to the culture 2 to 4 hours after electroporation, at theindicated MOI (1×10⁵ to 1×10⁶ range). Subsequently, edited cells werecultured using standard conditions (37° C. and expanded in T cell growthmedium, replenished as needed to maintain a density of ˜1×10⁶ cells/mlevery 2 to 3 days).

To obtain TCR-negative T cells, TCR-positive T cells were removed fromthe culture using magnetic biotin-anti-TCRαβ and anti-biotin microbeadsand LS columns (Miltenyi Biotech). For details of targeting constructsand strategies, see FIGS. 7 and 14.

Retroviral vector constructs, retroviral production and transduction:Plasmids encoding the SFG γ-retroviral (RV) vector (Riviàre et al.,Proc. Natl. Acad. Sci. USA 92:6733-6737 (1995)) were prepared aspreviously described (Brentjens et al., Nat. Med. 9, 279-286,(2003);Maher et al., Nat. Biotechnol. 20:70-75 (2002)). VSV-G pseudotypedretroviral supernatants derived from transduced gpg29 fibroblasts (H29)were used to construct stable retroviral-producing cell lines aspreviously described (Gong et al., Cancer patient T cells geneticallytargeted to prostate-specific membrane antigen specifically lyseprostate cancer cells and release cytokines in response toprostate-specific membrane antigen. Neoplasia 1:123-127 (1999)). T cellswere transduced by centrifugation on Retronectin (Takara)-coated plates.

Cell lines: NALM-6 and NIH/3T3 were obtained from ATCC and wereregularly tested for mycoplasma contamination using the MycoAlertMycoplasma Detection Kit (Lonza). NALM-6 cells were transduced toexpress firefly luciferase-GFP and NIH/3T3 cells transduced to expresshuman CD19 (Brentjens et al., Nat. Med. 9, 279-286, (2003); Zhao et al.,Cancer Cell 28:415-428 (2015)).

Cytotoxicity assays: The cytotoxicity of T cells transduced with a CARwas determined by standard luciferase-based assay. In brief, NALM-6expressing firefly luciferase-GFP served as target cells. The effector(E) and tumour target (T) cells were co-cultured in triplicates at theindicated E/T ratio using black-walled 96 well plates with 1×10⁵ targetcells in a total volume of 100 μl/well in NALM-6 Medium. Target cellsalone were plated at the same cell density to determine the maximalluciferase expression (relative light units; RLUmax). 18 hr later, 100μl luciferase substrate (Bright-Glo, Promega; Madison, Wis.) wasdirectly added to each well. Emitted light was detected in aluminescence plate reader or Xenogen IVIS Imaging System (Xenogen;Alameda, Calif.), and quantified using Living Image software (Xenogen).Lysis was determined as [1−(RLUsample)/(RLUmax)]×100.

Antigen stimulation and proliferation assays: NIH/3T3 expressing humanCD19 were used as artificial antigen-presenting cells (Brentjens et al.,Nat. Med. 9, 279-286, (2003)). For weekly stimulations, 3×105 IrradiatedCD19+ AAPCs were plated in 24 well plates 12 hours before the additionof 5×10⁵ CART cells in X-vivo 15 + human serum +50U IL-2/mL. Every 2days, cells were counted and media was added to reach a concentration of1×10⁶ T cells/mL. For repeated proximal stimulations (FIG. 6D), cellswere transferred to a new well plated with 3T3-CD19 after 24 h (2stimulations) or every 12 h (4 stimulations). For each condition, Tcells were counted and analysed by FACS for CAR, phenotypic andexhaustion markers expression every 12 h.

Antibodies and intracellular staining: CAR was labelled with a goatanti-mouse Fab (Jackson ImmunoResearch, 115-606-003; West Grove, Pa.).For T cell phenotyping, the following antibodies were used: mouseanti-human BUV-395CD4 (563552), APC-cy7-CD8 (557834), BV-421-CD62L(563862), BV-510-CD279 (PD1, 563076) from BD biosciences (San Jose,Calif.); mouse anti-human APC-CD25 (17-0259-42), FITC-CD45RA(11-0458-42), PerCP-eFluor710 CD223 (LAG-3, 46-2239-42) formeBiosciences (Carslbad, Calif.), and FITC mouse anti-human CD366 (TIM-3,345032) from Biolegend (San Diego, Calif.). For intracellular staining,T cells were fixed and permeabilized using BD Cytofix/Cytoperm Plus kit(BD Biosciences) as per the recommendation of the manufacturer.Anti-CD8-FITC (clone HIT8a, ebiosciencce) and anti-CD4-BUV-395 (cloneSK3, BD Horizon; BD Biosciences) were used for extracellular staining.Anti TNF-Alexa Fluor 700 (clone MAb 11, BD pharmingen; BD Biosciences),anti-IL2-BV421 (clone 5344.111, BD Horizon) and anti-IFNg-BV510 (cloneB27, BD Horizon) were used for intracellular staining.

Mouse Systemic Tumour Model: 8- to 12-week-old NOD/SCID/IL-2Rγ-null(NSG) male mice (Jackson Laboratory) were used, under a protocolapproved by the MSKCC Institutional Animal Care and Use Committee. Micewere inoculated with 0.5×10⁶FFLuc-GFP NALM-6 cells by tail veininjection, followed by 2×10⁵, 1×10⁵ or 5×10⁴, CART cells injected fourdays later. NALM-6 produce very even tumour burdens and no mice wereexcluded prior to treatment. No randomization or blinding methods wereused. Bioluminescence imaging utilized the Xenogen IVIS Imaging System(Xenogen) with Living Image software (Xenogen) for acquisition ofimaging datasets. Tumour burden was assessed as previously described(Gade et al., Cancer Res. 65:9080-9088 (2005)).

RNA extraction and real-time quantitative PCR: Total RNA was extractedfrom T cells by using the RNeasy kit (QIAGEN; Hilden, Germany) combinedwith QlAshredder (QIAGEN), following the manufacturer's instructions.RNA concentration and quality were assessed by UV spectroscopy using theNanoDrop spectrophotometer (Thermo Fisher Scientific; Carslbad, Calif.).One hundred to 200 ng total RNA were used to prepare cDNA using theSuperScript III First-Strand Synthesis SuperMix (Invitrogen; Carlsbad,Calif.), with a 1:1 volume ratio of random hexamers and oligo dT.Completed cDNA synthesis reactions were treated with 2U RNase H for 20min at 37° C. Quantitative PCR was performed using the ABsolute BlueqPCR SYBR Green Low ROX Mix (Thermo Fisher Scientific), and thefollowing primer sets: Ribosomal 18S: forward 5′-aacccgttgaaccccatt (SEQID NO:29), reverse 5′-ccatccaatcggtagtagcg (SEQ ID NO:30); 1928z:forward 5′-cgtgcagtctaaagacttgg (SEQ ID NO:31), reverse5′-ataggggacttggacaaagg (SEQ ID NO:32); T-bet: forward5′-gaaacccagttcattgccgt (SEQ ID NO:33), reverse 5′-ccccaaggaattgacagttg(SEQ ID NO:34); EOMES: forward 5′-actggttcccactggatgag (SEQ ID NO:35),reverse 5′-ccacgccatcctctgtaact (SEQ ID NO:36); GATA3: forward5′-cacaaccacactctggagga (SEQ ID NO:37), reverse 5′-ggtttctggtctggatgcct(SEQ ID NO:38). PCR assays were run on the QuantStudio™ 7 Flex System(Thermo Fisher Scientific), and Ct values were obtained with theQuantStudio Real-Time PCR software. Relative changes in gene expressionwere analysed with the 2^(ΔΔCt) method. RNA expression levels werenormalized to the percentage of CAR+ T cells for each group of T cellsanalysed.

Statistics: All experimental data are presented as mean±s.e.m. Nostatistical methods were used to predetermine sample size. Groups werecompared using the Welch's two-sample t-test for parametric data (samplesize>10) or the Mann-Whitney Test for non-parametric data (samplesize<10). Welch's correction was used, as the variances were not equal.For the comparison of CAR MFI and RNA level upon CAR stimulation, ANOVAF-tests were used. Statistical analysis was performed on GraphPad Prism7 software (GraphPad Software; La Jolla, Calif.).

To disrupt the TRAC locus and place the 1928z CAR (Brentj ens et al.,Sci. Transl. Med. 5, 177ra138 (2013)) under its transcriptional control(TRAC-CAR), a guide RNA was designed targeting the 5′ end of TRAC'sfirst exon and an adeno-associated virus (AAV) vector repair matrixencoding a self-cleaving P2A peptide followed by the CAR cDNA (FIG. 3Aand FIG. 7A). T cell electroporation of Cas9 mRNA and gRNA yielded ahigh knock-out (KO) frequency (˜70%, FIG. 3B and FIG. 7D) withoutlimited cell death. The knock-in (KI) was proportional to AAV dosage,exceeding 40% at a multiplicity of infection (MOI) of 106 (FIG. 3B andFIGS. 7C and 7E). This efficient targeting, reported here for the firsttime at the TRAC locus, is comparable to levels reached in T cells atthe AAVS1, CCR5 or CD40L loci (Sather et al., Sci. Transl. Med.7:307ra156 (2015); Wang et al., Nucleic Acids Res. 44:e30 (2016);Hubbard et al., Blood 127:2513-2522 (2016)). Approximately 95% of CAR+cells were T-cell receptor (TCR)—negative (FIG. 7G), validating the2-in-1 TCR-knockout and CAR-knock-in strategy. The observed 5% ofCAR+/TCR+ cells is consistent with the typical frequency ofdual-TCRα-expressing T cells (Corthay et al., J. Autoimmun. 16:423-429(2001)). The targeting specificity was confirmed by mapping AAV vectorintegration over the whole genome (de Vree et al., Nat. Biotechnol.32:1019-1025 (2014)), which confirmed the high selectivity for TRACintegration and absence of off-target hotspots (FIG. 8). These resultsdemonstrate the high efficiency and precision of gene targeting offeredby CRISPR/Cas9 and our ability to reproducibly generate up to 50×10⁶ ofTRAC-CAR T cells. Homogenous and consistent expression of TRAC-CAR wasfound in multiple donors, in contrast to retrovirally encoded CAR(RV-CAR), which showed variegated expression with a twofold higher meanexpression (FIGS. 3C and 3D).

In vitro functional studies did not reveal any notable differencesbetween TRAC-encoded and randomly integrated 1928z, in terms of eithercytotoxicity or T cell proliferation in response to weekly stimulationwith CD19+ antigen-presenting cells (Brentj ens et al., Nat. Med.9:279-286 (2003)) (FIGS. 9A and 9C). These experiments included acontrol group where TCR-disrupted T cells expressing retrovirallytransduced CAR (RV-CAR-TCR−) responded similarly to RV-CAR TCR+ T cells(FIG. 9A). In vivo, however, in the pre-B acute lymphoblastic leukaemiaNALM-6 mouse model using the “CAR stress test”, in which CAR T-celldosage is gradually lowered to reveal the functional limits of differentT-cell populations (Brentjens et al., Nat. Med. 9:279-286 (2003); Zhaoet al., Cancer Cell 28:415-428 (2015)), TRAC-CAR, RV-CAR and RV-CAR-TCR−T cells differed markedly in their anti-tumour activity. TRAC-CAR Tcells induced far greater responses and markedly prolonged mediansurvival at every T-cell dose (FIG. 3E and FIG. 10A). TCR disruption hadno discernable effect on the potency of RV-CAR T cells. Bone marrowstudies in mice injected with 1×10⁵ CAR T cells showed similar T-cellaccumulation at the tumour site after 10 days (FIG. 3F). However, onlythe TRAC-CAR T cells achieved tumour control (FIGS. 3G and 3H). By day17, TRAC-CAR T cells exceeded RV-CAR T cells in number, as the latterdiminished relative to day 10, despite the continued presence of CD19+tumour cells (FIG. 3F-3G and FIG. 10B). Furthermore, the CAR T-cellgroups differed in their degree of T-cell differentiation andexhaustion, as reflected in the proportion of terminal effector cells(CD45RA+CD62L−) and accumulation of co-expressed PD1, LAG3 and TIM3(Blackburn et al., Nat. Immunol. 10:29-37 (2009)), respectively. Thus,conventional CAR T cells showed up to 50% positive expression of themarkers of exhaustion by day 17, in contrast to less than 2% of theTRAC-CAR T cells, which also retained a larger effector memorycomposition (FIGS. 3I-3J and FIGS. 10C-10D). Terminal differentiationand acquisition of this exhaustion phenotype is consistent withdiminished anti-tumour activity (Gattinoni et al., Nat. Med.17:1290-1297 (2011)). Intriguingly, CAR expression in bone marrow Tcells was similar to pre-infusion levels for TRAC-CAR T cells butdiminished in both RV-CAR groups (FIG. 10E). Importantly, cell-surfaceexpression of the mutant LNGFR reporter (Gallardo et al., Gene Ther.4:1115-1119 (1997)) (co-expressed via a self-cleaving 2A element) wasundiminished, ruling out vector silencing as the explanation fordiminished CAR expression (FIGS. 10G-10H). The CAR expression levelmeasured in RV-CAR T cells negatively correlated with tumour burden(FIG. 10I), suggesting that cell-surface CAR was down-regulated inproportion to tumour antigen. These in vivo findings thus not onlydemonstrated the superior anti-tumour activity of TRAC-CAR T cells, butalso forged a link between tumour control, T cell differentiation andexhaustion, and CAR expression levels. These same patterns were observedwith another CAR, 19BBz, which utilizes the 4-1BB cytoplasmic domain asits costimulatory moiety (FIG. 11).

To further analyse the impact of CAR expression levels on T-cellfunction, we first examined T-cell phenotype when cultured in theabsence or presence of antigen (FIG. 4). Five days after transduction,RV-CAR T cells already showed evidence of activation, exhaustion anddifferentiation (FIG. 4A and FIG. 12A), similar to results obtained witha previously described retrovirally delivered CAR22. By contrast,TRAC-CAR T cells maintained a phenotype analogous to untransduced Tcells (FIG. 4A), mainly composed of naive and central memory cells(CD62L+ cells), a phenotype associated with greater in vivo anti-tumouractivity (Gattinoni et al., Nat. Med. 17:1290-1297 (2011); Sommermeyeret al., Leukemia 30:492-500 (2016)). Consistent with constitutiveactivating signalling, we found that RV-CARs, but not TRAC-CARs, hadphosphorylated immune-based tyrosine activation motifs (Long et al.,Nat. Med. 21:581-590 (2015)) (FIGS. 4B and 4C). Further differences werenoted upon exposure to antigen. In contrast to TRAC-CAR T cells, RV-CART cells stimulated 1, 2 or 4 times in a 48 h period differentiate intoeffector T cells, identified on the basis of phenotype (loss of CD62L),cytokine secretion (increased IFNγ, IL2 and TNFα) and expression ofmaster transcription factors (increased T-bet, EOMES and GATA3) (FIGS.4D-4E and FIGS. 12B-12D). These results indicated that the improvedefficacy of TRAC-CAR T cells is related to its CAR expression level byreducing tonic signalling and delaying T cell differentiation uponstimulation.

To control CAR expression, it was first attempted to vary the retroviralvector copy number. Lowered gene transfer efficiency only modestlyaffected the CAR expression level (FIG. 13). Interestingly, even whenmean RV-CAR expression matched that of TRAC-CAR, the former stilldisplayed accelerated differentiation upon multiple stimulations,suggesting that dynamic regulation of CAR expression, and not justbaseline expression, promotes distinct functional characteristics.

To further define the importance of CAR expression levels, T cells thatexpressed CAR from different genomic loci and promoters were generated.To examine the specific contribution of the TRAC locus and its promoter,a further seven constructs were designed targeting the 1928z CAR to theTRAC or the β2-microglobulin (B2M) locus (MHC-I related gene known to beexpressed in all T cells), using either endogenous or exogenouspromoters (FIGS. 5A-5B and FIGS. 14A-14E). Engineered CAR T cells weresuccessfully engineered at both loci, achieving homogenous CARexpression with mean levels ranging from seven times lower (B2M-PGK100)to more than double (TRAC-EF1α) of TRAC-CAR endogenous promoter (FIGS.5C-5E and FIG. 14).

All of the combinations that conferred higher CAR expression thanTRAC-CAR displayed the tonic signalling signature, in stark contrast tothose providing lower expression, consistent with a previous studylinking expression level to antigen-independent signalling (Frigault etal., Cancer Immunol Res. 3:356-367 (2015)) (FIG. 5E and FIG. 14F). Threeof these were selected for in depth analysis: high-expressing TRAC-EF1αand low-expressing B2M-CAR and TRAC-LTR (RV enhancer-promoter),comparing their in vitro and in vivo potency against TRAC-CAR. In vitro,following repeated antigenic stimulations, TRAC-EF1α CAR T cells rapidlyacquired effector profiles while B2M and TRAC-CAR T cells retained acentral memory phenotype (FIG. 5F and FIG. 15A). Interestingly, althoughTRAC-LTR directed lower baseline CAR expression than RV-CAR and avertedthe tonic signalling, the LTR still promoted from within the TRAC locusthe same differentiation pattern as RV-CAR. In the NALM-6 stress testmodel, none of the 3 locus-promoter combination displayed the sameanti-tumour efficacy as TRAC-CAR (FIGS. 5G-5H). 10 and 17 days afterinfusion of 1×10⁵ CAR T cells, the number of CAR T cells accumulated inbone marrow was similar or higher than for TRAC-CAR T cells; however,only TRAC-CAR T cells could efficiently control tumour progression(FIGS. 15C-15E). Although B2M-CAR T cells seemed to preserve aneffector/effector-memory ratio similar to TRAC-CAR T cells, they tooacquired a preponderant exhaustion signature (FIGS. 15F-15G), suggestingthat delayed differentiation may be independent from exhaustion.Together these results underscored the effect of CAR targeting andfurther suggested regulation of CAR expression extending beyond baselinetranscriptional control.

CAR expression was closely analyzed upon encounter with antigen. To thisend, CAR T cells were admixed with CD19+ antigen-presenting cells andcell-surface CAR expression was examined at regular time intervals (FIG.6A). CAR expression decreased within hours of exposure to CD19 in bothtargeted and randomly integrated CAR T cells, accompanied by a deeperdrop and longer recovery lag when the initial level was lower. Thesubsequent return to baseline expression most notably distinguished thedifferent T-cell populations.

To better study the mechanism behind the drop in the CAR cell-surfaceexpression, we designed a CAR-GFP fusion protein to analyse bothcell-surface and intra-cellular CAR expression, and compare it to cellsexpressing a CAR with a co-translated but cleaved LNGFR reporter (FIGS.6B-6C). We observed that CAR expression was downregulated independentlyof LNGFR, suggesting a physical internalization rather than atranscriptional process. The co-reduction of CAR and GFP signalfollowing antigen encounter indicated that CAR internalization wasfollowed by its degradation. The occurrence of CAR degradation followingexposure to antigen suggested that de novo CAR synthesis from CAR mRNAwould be needed to precisely and timely restore CAR expression andsupport effective T-cell function. Careful analysis of CAR cell-surfaceexpression following repeated antigen stimulation (FIG. 6D and FIG. 16A)identified two main patterns in the recovery phase (12-48 h hours afterantigen exposure). In TRAC-EF1α, TRAC-LTR and RV-CAR T cells, CARcell-surface expression increased after each stimulation, two- tofourfold above baseline within 24 h. In both TRAC- and B2M-CAR T cells,CAR expression decreased upon repeated stimulations and remained belowbaseline after 48 hours (FIG. 6D). Steady-state mRNA analysis showed alinear correlation between cell-surface protein level (FIG. 6E and FIG.16B) and the transcriptional response to CAR T cell activation (FIG.6F), pointing to the essential role of promoter strength and regulationto enable optimal post-stimulation replenishment of cell-surface CARexpression.

This CAR protein/RNA downregulation and subsequent re-expression isreminiscent of TCR regulation upon stimulation of human T cells (Schrumet al., Immunol Rev. 196:7-24, (2003)) and antigen-induced TCRrecirculation in mouse T cells (Liu et al., Immunity 13, 665-675,(2000); Call et al., Annu. Rev. Immunol. 23, 101-125, (2005); Allison etal., Elife. 5,(2016)). Similarly, accelerated differentiation andexhaustion have been reported in the context of excessive and continuousactivation of the TCR (Schietinger et al., Trends Immunol. 35, 51-60,(2014); Wherry et al., Nat. Rev. Immunol. 15, 486-499, (2015)).Altogether, these converging findings support the conclusion that TRAChas a role in control of CAR expression in two critical ways. One is topromote optimal baseline expression, which prevented tonic signalling inthe absence of antigen and allowed effective CAR internalization uponsingle or multiple contacts with antigen. The other is to direct abalanced transcriptional response resulting in a kinetically optimalrecovery of baseline CAR expression after antigen engagement. Incontrast to T cells with higher CAR expression, the TRAC-CAR profilecorrelated with decreased T-cell differentiation and exhaustion,resulting in superior tumour eradication. Our studies, which comparedrandomly integrating CARs versus CARs targeted to two loci in 8different transcriptional configurations, illustrate the exquisitesensitivity of CAR regulation. Thus, although the endogenous B2Mpromoter responded similarly to TRAC upon CAR stimulation, B2M-CAR didnot perform as well as TRAC-CAR in vivo, indicating that the lower basalexpression level it offered is insufficient for effective CAR activity.TRAC-LTR likewise provided baseline expression comparable to TRAC, butits prompt rebound after activation was associated with poor T-cellperformance and accelerated differentiation. We therefore conclude thatboth the basal and dynamic CAR expression levels contribute tosustaining T-cell function.

In summary, the results demonstrate that targeting a CAR coding sequenceto the TCR locus, placing it under the control of endogenous regulatoryelements, reduces tonic signalling, averts accelerated T-celldifferentiation and exhaustion, and increases the therapeutic potency ofengineered T cells. The kinetic measurements of antigen-induced CARinternalization and degradation revealed differential recovery ofcell-surface CAR depending on the enhancer/promoter elements driving CARexpression. These findings demonstrate that tight transcriptionalregulation of CAR expression is critical for effective tumoureradication. The targeting of CARs to a TCR locus may thus provide asafer therapeutic T cell (by minimizing the risks of insertionaloncogenesis and TCR-induced autoimmunity and alloreactivity), a betterdefined T-cell product (by yielding constant CAR expression and avoidingposition-effect variegation and vector copy number variation) and a morepotent T cell (by reducing constitutive signalling and delaying T-cellexhaustion). Finally, the results demonstrate the relevance of studyingCAR immunobiology and the vast potential of genome editing to advanceT-cell therapies.

8.3 Example 3 Expression of Therapeutic Transgenes Under Control ofEndogenous Promoters

In one example, a therapeutic chimeric antigen receptor (CAR) isintegrated at the TRAC locus (under the control of endogenouspromoter/enhancer elements); an NFAT-responsive transcription unit isintegrated at the T cell receptor beta chain constant (TRBC) locus, fromwhich two therapeutic PD1L-specific and-CTLA4 scFvs are expressed. Withthis setup, engineered T cells are activated through the CAR, whichleads to NFAT activation followed by the expression of the therapeuticscFvs. Alternatively, these transgenes can be integrated at theNFAT-responsive CD69 locus. In a particular embodiment, a chimeric cellsurface ligand-transcription factor is CD19-NFAT. Accordingly, in oneembodiment, the CAR is encoded by a first transgene, and the PD1L scFvand the CTLA4 scFV are encoded by a second transgene that isbicistronic, wherein the expression of the second transgene is under thecontrol of the endogenous TRBC promoter that is induced by NFAT. In analternative embodiment, the PD1L and CTLA4 scFvs are expressed fromseparate transgenes (i.e., second and third transgenes). In a specificembodiment, the PD1L and CTLA4 scFvs are expressed from a single,polycistronic transgene. Such a construct can optionally include acleavable sequence, such as a P2A sequence, to provide for expression ofthe PD1L and CTLA4 scFvs as separate molecules.

In another example, a chimeric cell-surface ligand(extracellular)-transcription factor (TF; intracellular) fusion gene isintegrated at the TRAC locus (under the control of endogenouspromoter/enhancer elements) that specifically interacts with the CD19molecule in B cells; a TF-responsive transcription unit integrated atthe TRBC locus, from which a therapeutic chimeric immune receptor ligand(CIRL) is expressed. This design allows engineered T cells to respond tothe interaction with B cells by releasing the TF, which then activatesthe expression of the CIRL, which interacts with a specific autoimmuneB-cell immunoglobulin receptor (IgR). The latter interactionsignals/activates a cytotoxic T-cell response leading to autoimmuneB-cell death. In one embodiment, a chimeric cell-surface ligand(extracellular)-transcription factor (TF; intracellular) fusion gene isencoded by a first transgene. In one embodiment, the CIRL is encoded bya second transgene.

In another example, a DNA sequence encoding a HIV-specific ribozyme isintegrated at CD4 locus; an interferon-responsive transcription unitintegrated at the CCR5 locus that expresses an intracellular scFv thatinteracts with HIV Rev protein. This therapeutic T cell will inhibit HIVreplication threefold: by cleaving the HIV genome through the ribozyme,preventing HIV infection by eliminating CCR5 expression, and inhibitingHIV packaging by blocking HIV Rev activity. In one embodiment, anHIV-specific ribozyme is encoded by a first transgene. In oneembodiment, an intracellular scFv is encoded by a second transgene, forexample, a scFv that interacts with HIV rev protein.

8.4 Example 4 Generation of Non-Integrating Gamma-Retrovirus

Recombinant non-integrating (or integration-deficient) gamma-retrovirus(rNIgRV or IDgRV) is a retroviral vector that contains a mutantintegrase protein, which cannot catalyze viral DNA integration into thehost cell genome. To make this mutant retroviral vector, a plasmid DNAencoding a mutant integrase protein (with mutations as indicatedpreviously) is used in combination with the envelope-encoding and theretroviral genome-encoding plasmid DNAs. These three plasmids aretransfected into producer mammalian cells, and the recombinant mutantviral vector is released into the medium, which is later collected andused to transduce human peripheral blood T cells.

As shown in FIG. 18, mutant integrases were generated by mutating aminoacids in the DDE motif (see Andrake and Skalka (2015). RetroviralIntegrase: Then and Now. Ann. Rev. Virol. 2:241-264. The DDE amino acidspositions are: D124, D183, E 219 (residue numbering based on GenBankaccession number NP_955592 (NP_955592.1). The mutants generated wereD124A, D124E, D124N, D124V, D183A, D183N, D124A and D183A, D124A andD183N, D124E and D183A, D124E and D183N, D124N and D183A, D124N andD183N, D124V and D183A, and D124V and D183N. Mutants were generatedusing standard molecular biology techniques. A plasmid containing theMoloney Murine Leukemia Virus (MLV) Gag-Pol sequences was modified usingstandard molecular techniques. Mutants were generated by replacing theDNA sequence region containing the DDE motif with a new DNA sequencewhere the specific codon(s) is mutated to generate the each specificmutant. The resulting plasmids were used to produce NIgRVs.

Taking advantage of its transient nature inside the target cells,rNIgRVs can be used for different applications. For example, totransiently express genes that maintain a certain cell phenotype -likememory T cells; to transiently expresses chimeric nucleases orCRISPR/Cas components to disrupt specific DNA sequences; to deliverexogenous DNA flanked with DNA sequences homologous to specific genomiclocations, thus enabling integration of the flanked DNA sequence intothe T cell genome via homologous recombination; to deliver and integratethe transgenic retroviral DNA at specific DNA breaks in anintegrase-independent NHEJ-dependent manner.

9. REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A T cell wherein a transgene is integrated at afirst site within the genome of the T cell such that expression of thetransgene is under control of an endogenous promoter of the T cell,wherein the transgene encodes a therapeutic protein or therapeuticnucleic acid.
 2. A T cell wherein a first transgene is integrated at afirst site within the genome of the T cell such that expression of thefirst transgene is under control of a first endogenous promoter of the Tcell, and wherein a second transgene is integrated at a second sitewithin the genome of the T cell, such that expression of the secondtransgene is under the control of a second endogenous promoter, whereinsaid first and second endogenous promoters are different promoters, andwherein the first transgene encodes a first therapeutic protein or firsttherapeutic nucleic acid, and the second transgene encodes a secondtherapeutic protein or second therapeutic nucleic acid, preferablywherein the first therapeutic protein or first therapeutic nucleic acidis different from said second therapeutic protein or second therapeuticnucleic nucleic, respectively.
 3. The T cell of claim 1 or 2, whereinthe T cell is an immunostimulatory T cell.
 4. The T cell of claim 1 or2, wherein the T cell is an immunoinhibitory T cell.
 5. An isolatedpopulation of T cells, which comprises a plurality of the T cell ofclaim 1 or
 2. 6. An isolated population of T cells, which comprises aplurality of the T cell of claim
 3. 7. An isolated population of Tcells, which comprises a plurality of the T cell of claim
 4. 8. Apharmaceutical composition comprising a therapeutically effective amountof the T cell of claim 1 or 2; and a pharmaceutically acceptablecarrier.
 9. A pharmaceutical composition comprising a therapeuticallyeffective amount of a population of T cells, which population comprisesa plurality of the T cell of claim 1 or 2; and a pharmaceuticallyacceptable carrier.
 10. A pharmaceutical composition comprising atherapeutically effective amount of the T cell of claim 3; and apharmaceutically acceptable carrier.
 11. A pharmaceutical compositioncomprising a therapeutically effective amount of a population of Tcells, which population comprises a plurality of the T cell of claim 3;and a pharmaceutically acceptable carrier.
 12. A pharmaceuticalcomposition comprising a therapeutically effective amount of the T cellof claim 4; and a pharmaceutically acceptable carrier.
 13. Apharmaceutical composition comprising a therapeutically effective amountof a population of T cells, which population comprises a plurality ofthe T cell of claim 4; and a pharmaceutically acceptable carrier.
 14. Amethod of treating a subject with T cell therapy in need thereof,comprising administering to the subject a therapeutically effectiveamount of the T cell of claim 1 or
 2. 15. A method of treating a subjectwith T cell therapy in need thereof, comprising administering to thesubject a therapeutically effective amount of the T cell population ofclaim
 5. 16. A method of treating a subject with T cell therapy in needthereof, comprising administering to the subject the pharmaceuticalcomposition of claim 8 or
 9. 17. A method of treating a subject with Tcell therapy in need thereof, wherein the subject is in need of astimulated immune response, comprising administering to the subject atherapeutically effective amount of a cell or population of cells,wherein the cell is a T cell, wherein a transgene is integrated at afirst site within the genome of the T cell such that expression of thetransgene is under control of an endogenous promoter of the T cell,wherein the transgene encodes a therapeutic protein or therapeuticnucleic acid.
 18. The T cell of claim 17, wherein the T cell is animmunostimulatory T cell.
 19. The T cell of claim 18, wherein the T cellis selected from the group consisting of cytotoxic T lymphocyte (CTL),CD4+ subtype, CD8+ subtype, central memory T cell (TCM), stem memory Tcell (TSCM), effector memory T cell, effector T cell, Th1 cell, Th2cell, Th9 cell, Th17 cell, Th22 cell, and Tfh (follicular helper) cell.20. A method of treating a subject with T cell therapy in need thereof,wherein the subject is in need of an inhibited immune response,comprising administering to the subject a therapeutically effectiveamount of a cell or population of cells, wherein the cell is a T cell,wherein a transgene is integrated at a first site within the genome ofthe T cell such that expression of the transgene is under control of anendogenous promoter of the T cell, wherein the transgene encodes atherapeutic protein or therapeutic nucleic acid.
 21. The method of claim20, wherein the T cell is an immunoinhibitory T cell.
 22. The method ofclaim 21, wherein the T cell is a regulatory T cell.
 23. A method ofgenerating a T cell that expresses a therapeutic transgene, comprising:introducing into a T cell: (i) a transgene, and (ii) a homologousrecombination system suitable for targeted integration of the transgeneat a site within the genome of the cell, whereby the homologousrecombination system integrates the transgene at said site within thegenome of the cell, and wherein expression of the transgene is under thecontrol of an endogenous promoter, wherein the transgene encodes atherapeutic protein or a therapeutic nucleic acid.
 24. A vectorcomprising a non-integrating gamma-retrovirus.
 25. The vector of claim24, wherein the non-integrating gamma-retrovirus comprises a mutatedintegrase.
 26. A T cell wherein a recombinant nucleic acid sequenceencoding a chimeric antigen receptor (CAR) is integrated at a first sitewithin the genome of the cell such that the CAR is expressed by the cellat the surface of the cell, and wherein integration of the nucleic acidencoding the CAR at said first site reduces or prevents expression of afunctional T cell receptor (TCR) complex at the surface of the cell. 27.A human T cell wherein a promotor-less recombinant nucleic acid sequenceencoding a CAR is integrated at a site in the genome of the cell, saidsite being the first exon of the TCR alpha chain, such that the CAR isexpressed under control of the endogenous TCR alpha chain promoter, toproduce said CAR at the surface of the cell, and wherein integration ofthe CAR at said site reduces or prevents expression of a functional TCRalpha chain.
 28. An isolated population of T cells, which comprises aplurality of the cell of claim 26 or
 27. 29. A pharmaceuticalcomposition comprising a therapeutically effective amount of the cell ofclaim 26 or 27; and a pharmaceutically acceptable carrier.
 30. Apharmaceutical composition comprising a therapeutically effective amountof a population of T cells, which population comprises a plurality ofthe cell of claim 26 or 27; and a pharmaceutically acceptable carrier.31. A method of treating a subject with CAR therapy in need thereof,comprising administering to the subject a therapeutically effectiveamount of the cell of claim 26 or
 27. 32. A method of treating a subjectwith CAR therapy in need thereof, comprising administering to thesubject the pharmaceutical composition of claim
 29. 33. A method oftreating a subject with CAR therapy in need thereof, comprisingadministering to the subject a therapeutically effective amount of thecell population of claim
 28. 34. A method of treating a subject with CARtherapy in need thereof, comprising administering to the subject thepharmaceutical composition of claim
 30. 35. A method of generating a Tcell that expresses a chimeric antigen receptor (CAR) and lacks afunctional T cell receptor (TCR) complex, comprising: introducing into aT cell: (i) a nucleic acid sequence encoding a CAR, and (ii) ahomologous recombination system suitable for targeted integration of thenucleic acid sequence at a site within the genome of the cell, wherebythe homologous recombination system integrates the nucleic acid sequenceencoding the CAR at said site within the genome of the cell such thatintegration of the CAR at said site reduces or prevents expression of afunctional T cell receptor complex at the surface of the cell, therebygenerating a T cell that expresses the CAR and lacks a functional TCRcomplex.
 36. An induced pluripotent stem cell, wherein a recombinantnucleic acid sequence encoding a chimeric antigen receptor (CAR) isintegrated at a first site within the genome of the cell such that theCAR is expressed by the cell at the surface of the cell, and whereinintegration of the nucleic acid encoding the CAR at said first sitereduces or prevents expression of a functional T cell receptor (TCR)complex at the surface of the cell.